Method for the production and purification of adenoviral vectors

ABSTRACT

The present invention addresses the need to improve the yields of viral vectors when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsCl banding using an ultracentrifuge.

The present application is a divisional of co-pending application Ser.No. 09/203,078 filed Dec. 1, 1998, which was a continuation in-part ofco-pending U.S. patent application Ser. No. 08/975,519 filed Nov. 29,1997 which is based on U.S. Provisional Patent Application Ser. No.60/031,329 filed Nov. 20, 1996.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of cell cultureand virus production. More particularly, it concerns improved methodsfor the culturing of mammalian cells, infection of those cells withadenovirus and the production of infectious adenovirus particlestherefrom.

2. Description of Related Art

Adenoviral vectors, which carry transgenes that can be transcribed andtranslated to express therapeutic proteins, are currently beingevaluated in the clinic for the treatment of a variety of cancerindications, including lung and head and neck cancers. As the clinicaltrials progress, the demand for clinical grade adenoviral vectors isincreasing dramatically. The projected annual demand for a 300 patientclinical trial could reach approximately 1.08×10¹⁶ viral particles.

Traditionally, adenoviruses are produced in commercially availabletissue culture flasks, “cellfactories,” or RB. Virus infected cells areharvested and subjected to multiple freeze-thaws to release the virusesfrom the cells in the form of crude cell lysate. The produced crude celllysate (CCL) is then purified by multiple CsCl gradientultracentrifugation steps. The typically reported virus yield from 100single tray cellfactories is about 1×10¹⁴ viral particles. Clearly, itbecomes unfeasible to produce the required amount of virus using thistraditional process. New scaleable and validatable production andpurification processes have to be developed to meet the increasingdemand.

The purification throughput of CsCl gradient ultracentrifugation is solimited that it cannot meet the demand for adenoviral vectors for genetherapy applications. Therefore, in order to achieve large scaleadenoviral vector production, purification methods other than CsClgradient ultracentrifugation have to be developed. Reports on thechromatographic purification of viruses are very limited, despite thewide application of chromatography for the purification of recombinantproteins. Size exclusion, ion exchange and affinity chromatography havebeen evaluated for the purification of retroviruses, tick-borneencephalitis virus, and plant viruses with varying degrees of success(Crooks, et al., 1990; Aboud, et al., 1982; McGrath et al., 1978, Smithand Lee, 1978; O'Neil and Balkovic, 1993). Even less research has beendone on the chromatographic purification of adenovirus. This lack ofresearch activity may be partially attributable to the existence of theeffective, albeit non-scalable, CsCl gradient ultracentrifugationpurification method for adenoviruses.

Recently, Huyghe et al. (1996) reported adenoviral vector purificationusing ion exchange chromatography in conjunction with metal chelateaffinity chromatography. Virus purity similar to that from CsCl gradientultracentrifugation was reported. Unfortunately, only 23% of virus wasrecovered after the double column purification process. Process factorsthat contribute to this low virus recovery are the freeze/thaw steputilized by the authors to lyse cells in order to release the virus fromthe cells and the two column purification procedure.

Clearly, there is a demand for an effective and scaleable method ofadenoviral vector production that will result in a high yield of productto meet the ever increasing demand for such products. Recently Blancheet al in WO 98/00524, based on U.S. Ser. No. 60/026,667, describeadenoviral production methods that are useful as descriptive art. PCTpublication No. WO 98/00524 and U.S. Ser. No. 60/026,667 arespecifically herein incorporated by reference for their description oftechniques for production and purification of recombinant adenovirus.

SUMMARY OF THE INVENTION

The present invention describes a new large scale process for theproduction and purification of adenovirus. This new production processoffers not only scalability and validatability but also virus puritycomparable to that achieved using CsCl gradient ultracentrifugation.

The present invention relates to a process for preparing large scalequantities of adenovirus. Indeed, it is believed that very largequantities of adenovirus particles can be produced using the processesof the present invention, quantities of up to about 1×10¹⁸ particles,and preferably at least about 5×10¹⁴ particles. This is highlydesirable, as there are currently no techniques available to produce thevery large, commercial quantities of adenovirus particles required forclinical applications at the high level of purity needed.

In one embodiment, the process generally involves preparing a culture ofproducer cells by seeding producer cells into a culture medium,infecting cells in the culture after they have reached a mid-log phasegrowth with a selected adenovirus (e.g., a recombinant adenovirus), andharvesting the adenovirus particles from the cell culture. This isbecause it has surprisingly been discovered by the inventors thatmaximal virus production is achieved in the producer cells when they areinfected in the later part of log phase growth and prior to stationarygrowth. Preferably, the adenovirus particles so obtained are thensubjected to purification techniques either known in the art or setforth herein.

In certain preferred embodiments of the present invention, therefore,the producer cells are infected with adenovirus at between about mid-logphase and stationary phase of growth. The log phase of the growth curveis where the cells reach their maximum rate of cell division (i.e.growth). The term mid-log phase of growth refers to the transitionmid-point of a logarithmic growth curve. Stationary phase growth refersto the time on a growth curve (i.e. a plateau) in which cell growth andcell death have come to equilibrium.

In even more preferred embodiments, the producer cells are infected withthe adenovirus during or after late-log phase of growth and beforestationary phase. Late-log phase is defined as cell growth approachingthe end of logarithmic growth, and before reaching the stationary phaseof growth. Late-log phase can typically be identified on a growth curveas a secondary or tertiary point of inflection that occurs as thelog-growth phase slows, approaching stationary growth.

In a preferred embodiment of the present invention, the producer cellsare seeded into the cell culture medium using an essentially homogeneouspool of cells. The inventors have surprisingly discovered that the useof a homogeneous pool of cells for seeding can provide much improvedconfluency and cell density as well as better maturation of the virus,which in turn provides for larger production quantities and ultimatepurity of the virus recovered. Indeed, seeding through the use ofseparate rather than homogeneous cell populations, for example fromindividual cell culture devices used in the cell expansion phase, canresult in uneven cell density, and therefore uneven confluency levels atthe time of infection. It is believed that the use of a homogeneous cellpool for seeding overcomes these problems.

In another preferred embodiment of the present invention, the culturemedium is at least partially perfused during a portion of time duringcell growth of the producer cells or following infection. Perfusion isused in order to maintain desired levels of certain metabolites and toremove and thereby reduce impurities in the culture medium. Perfusionrates can be measured in various manners, such as in terms ofreplacement volumes/unit time or in terms of levels of certainmetabolites that are desired to be maintained during times of perfusion.Of course, it is typically the case that perfusion is not carried out atall times during culturing, etc., and is generally carried out only fromtime to time during culturing as desired. For example, perfusion is nottypically initiated until after certain media components such as glucosebegin to become exhausted and need to be replaced.

The inventors have discovered that low perfusion rates are particularlypreferred, in that low perfusion rates tend to improve one's ability toobtain highly purified virus particles. The inventors prefer to defineperfusion rate in terms of the glucose level that is achieved ormaintained by means of the perfusion. For example, in the presentinvention the glucose concentration in the medium is preferablymaintained at a concentration of between about 0.5 g/L and about 3.0g/L. In a more preferred embodiment, the glucose concentration ismaintained at between about 0.70 g/L and 2.0 g/L. In a still morepreferred embodiment, the glucose concentration is maintained at betweenabout 1.0 g/L and 1.5 g/L.

Also in certain preferred embodiments, the inventors prefer torecirculate the cell culture media while carrying out processes inaccordance with the present invention, and even more preferably, therecirculation is carried out continuously. Recirculation is desirable inthat it affords a more even distribution of nutrients throughout thecell growth chamber.

In certain other embodiments, the cells are seeded into the culturemedium and allowed to attach to a culture surface for between about 3hours and about 24 hours prior to initiation of medium recirculation.Attachment of cells to a cell surface generally allows for a moreconsistent and uniform cell growth and higher virus production rate,which in turn allows for the production of higher quality virus. It hasbeen found by the inventors that recirculation can sometimes impedeconsistent and uniform cell attachment, and that ceasing recirculationduring cell attachment phases can provide significant advantages.

With respect to seeding, in a preferred embodiment of the presentinvention, the cell culture medium is seeded with between about 0.5×10⁴and about 3×10⁴ cells/cm², and more preferably with from about 1–2×10⁴cells/cm². The reason for this is that it has been found that in orderto achieve maximal cell expansion and growth, it is most preferable toinoculate the selected growth chamber with a lower number of cells thatone might typically use in other cell growth situations. The inventorshave found that higher numbers of cells used in the cell inoculationstep results in a cell density that is too high and can result in anover-confluence of cells at the time of viral infection, thus loweringyields. It is well within one of skill in the art to determine that inother types of cell culturing systems, similar optimization of theseeding density for a particular system could easily be determined.Nevertheless, in a particularly preferred embodiment, the cell culturemedium is seeded with between about 7.5×10³ and about 2.0×10⁴ cell/cm².In an even more preferred embodiment, the cell culture medium is seededwith between about 9×10³ and 1.5×10⁴ cells/cm².

In another preferred embodiment of the present invention, the harvestedadenovirus is purified and placed in a pharmaceutically acceptablecomposition. A pharmaceutically acceptable composition is defined as onethat meets the minimal safety required set forth by the FDA or othersimilar pharmaceutical governing body, and can thus be administeredsafely to a patient. The present invention provides processes for thepurification of the adenovirus. For example, the adenovirus is purifiedby steps that include chromatographic separation. While more than onechromatography step can be used in accordance with the present inventionto purify the adenovirus, this will often result in significant lossesin terms of yield. Thus, the inventors have discovered that surprisinglevels of purity can be achieved where only a single chromatography stepis carried out, particularly where that chromatography step is carriedout using ion-exchange chromatography. Ion-exchange chromatography is anexcellent choice for purification of adenovirus particles due to thepresence of a net negative charge on the surface of adenoviruses atphysiological pH, permitting high purity isolation of adenovirusparticles.

In particular embodiments of the present invention, the recombinantadenovirus is a replication-deficient adenovirus encoding a therapeuticgene operably linked to a promoter. A replication deficient adenoviruscarrying a therapeutic gene linked to a promoter allows the controlledexpression of the therapeutic gene by activating the promoter. Theprecise choice of a promoter further allows tissue specific regulationand expression of the therapeutic gene. In particular embodiments, thepromoter is an SV40 IE, RSV LTR, β-actin, CMV-IE, adenovirus major late,polyoma F9-1, or tyrosinase promoter.

In other embodiments the replication deficient adenovirus is lacking atleast a portion of the E1 region of the adenoviral genome. Replicationdeficient adenoviruses lacking a portion of the E1 region are desired toreduce toxicity and immunologic reaction to host cells. In anotherembodiment of the present invention, the producer cells complement thegrowth of replication deficient adenoviruses. This is an importantfeature of producer cells required to maintain high viral particlenumber of the replication deficient adenovirus. In certain suchembodiments, the producer cells are 293, PER.C6, 911 or IT293SF cells.In a preferred embodiment, the producer cells are 293 cells. This allows

In a preferred embodiment of the present invention it is contemplatedthat the recombinant adenovirus encodes a therapeutic recombinant gene.For example, the therapeutic gene may encode antisense ras, antisensemyc, antisense raf antisense erb, antisense src, antisense fms,antisense jun, antisense trk, antisense ret, antisense gsp, antisensehst, antisense bcl antisense abl, Rb, CFTR, p16, p21, p27, p57, p73,C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF, mda-7, thymidinekinase or p53. In an even more preferred embodiment, the therapeuticgene is p53. One of the most frequent abnormalities resulting in humancancer are mutations in p53, thus the ability to replace a deficient p53gene using the present invention is highly desirable.

In another particular embodiment of the present invention, theadenovirus is harvested by steps that include lysing the producer cellsby means other than freeze-thaw. The reason for this is that thefreeze-thaw method is somewhat cumbersome and not particularly suited toproduction of commercial quantities. In preferred embodiments theproducer cells are lysed by means of detergent lysis or autolysis. Theharvesting of the adenovirus by detergent lysis and autolysis results ina much higher virus recovery than the freeze-thaw process and istherefore an improvement in the large scale production of adenoviruses.

In a particular embodiment of the present invention the purifiedrecombinant adenovirus has one or more of the following properties. Forexample, the property may be a virus titer of between about 1×10⁹ andabout 1×10¹³ pfu/ml, a virus particle concentration between about 1×10¹⁰and about 2×10¹³ particles/ml, a particle:pfu ratio between about 10 andabout 60, less than 50 ng BSA per 1×10¹² viral particles, between about50 pg and 1 ng of contaminating human DNA per 1×10¹² viral particles ora single HPLC elution peak consisting essentially of 97 to 99% of thearea under the peak. These criteria select for a highly purifiedadenovirus.

To further impose limits on the purification process of the adenovirus,between about 5×10¹⁴ and 1×10¹⁸ viral particles are desired. Inaddition, one or more of the following properties further improve theselection for high purity adenovirus particles. For example the propertymay be a virus titer of between about 1×10⁹ and about 1×10¹³ pfu/ml,more preferably 1×10¹¹ and about 1×10¹³ pfu/ml, and most preferably1×10¹² and about 1×10¹³ pfu/ml. Further, a virus particle concentrationbetween about 1×10¹⁰ and about 2×10¹³ particles/ml, more preferably1×10¹¹ and about 2×10¹³ particles/ml, and most preferably 1×10² andabout 1×10¹³ particles/ml.

Additionally, a particle:pfu ratio between about 10 and about 60, morepreferably a particle:pfu ratio between about 10 and about 50, even morepreferable a particle:pfu ratio between about 10 and about 40, and mostpreferably a particle:pfu ratio between about 20 and about 40.

To limit the BSA concentration, it is preferable to have less than 50 ngBSA per 1×10¹² viral particles, for example, between about 1 ng to 50 ngBSA per 1×10¹² viral particles, and more preferably between about 5 ngand 40 ng of BSA per 1×10¹² viral particles.

Low concentrations of DNA contamination are also desired. Thus, betweenabout 50 pg and 1 ng of contaminating human DNA per 1×10¹² viralparticles is acceptable, even more preferable is between about 50 pg and500 pg of contaminating human DNA per 1×10¹² viral particles, and mostpreferable is between about 100 pg and 500 pg of contaminating human DNAper 1×10¹² viral particles. Finally, an adenovirus that elutes as asingle HPLC peak is desired, more preferably is an adenovirus thatelutes as an HPLC peak that contains between about 97 and 99% of thetotal area under the peak.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A and FIG. 1B. HPLC profiles of the viral solutions fromproduction runs using medium perfusion rates characterized as “high”(FIG. 1A) and “low” (FIG. 1B).

FIG. 2. The HPLC profile of crude cell lysate (CCL) from CellCube™(solid line A₂₆₀; dotted line A₂₈₀).

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D and FIG. 3E. The HPLC profiles oflysis solutions from CellCube™ using different detergents. FIG. 3AThesit®. FIG. 3B Triton®X-100. FIG. 3C. NP40®. FIG. 3D. Brij®80. FIG.3E. Tween®20. Detergent concentration: 1% (w/v) lysis temperature: roomtemperature. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 4A and FIG. 4B. The HPLC profiles of virus solution before (FIG.4A) and after (FIG. 4B) Benzonase treatment. (solid line A₂₆₀; dottedline A₂₈₀).

FIG. 5. The HPLC profile of virus solution after Benzonase treatment inthe presence of 1M NaCl. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 6. Purification of AdCMVp53 virus under buffer A condition of 20 mMTris+1 mM MgCl₂+0.2M NaCl, pH=7.5.

FIG. 7. Purification of AdCMVp53 virus under buffer A condition of 20 mMTris+1 mM MgCl₂+0.2M NaCl, pH=9.0.

FIG. 8A, FIG. 8B, and FIG. 8C. HPLC analysis of fractions obtainedduring purification FIG. 8A fraction 3. FIG. 8B fraction 4, FIG. 8Cfraction 8. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 9. Purification of AdCMVp53 virus under buffer A condition of 20 mMTris+1 mM MgCl₂+0.3M NaCl, pH=9.

FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D and FIG. 10E. HPLC analysis ofcrude virus fractions obtained during purification and CsCl gradientpurified virus. FIG. 10A Crude virus solution. FIG. 10B Flow through.FIG. 10C. Peak number 1. FIG. 10D. Peak number 2. FIG. 10E. CsClpurified virus. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 11. HPLC purification profile from a 5 cm id column.

FIG. 12. The major adenovirus structure proteins detected on SDS-PAGE.

FIG. 13. The BSA concentration in the purified virus as detected levelof the western blot assay.

FIG. 14. The chromatogram for the crude cell lysate material generatedfrom the CellCube™.

FIG. 15. The elution profile of treated virus solution purified usingthe method of the present invention using Toyopearl SuperQ resin.

FIG. 16A and FIG. 16B. HPLC analysis of virus fraction from purificationprotocol. FIG. 16A HPLC profiles of virus fraction from firstpurification step. FIG. 16B HPLC profiles of virus fraction from secondpurification. (solid line A₂₆₀; dotted line A₂₈₀).

FIG. 17. Purification of 1% Tween® harvest virus solution under lowmedium perfusion rate.

FIG. 18. HPLC analysis of the virus fraction produced under low mediumperfusion rate.

FIG. 19A, FIG. 19B and FIG. 19C. Analysis of column purified virus. FIG.19A SDS-PAGE analysis. FIG. 19B Western blot for BSA. FIG. 19C nucleicacid slot blot to determine the contaminating nucleic acidconcentration.

FIG. 20A, FIG. 20B, FIG. 20C, FIG. 20D, FIG. 20E and FIG. 20F. Capacitystudy of the Toyopearl SuperQ 650M resin. FIG. 20A Flow through fromloading ratio of 1:1. FIG. 20B. Purified virus from loading ratio of1:1. FIG. 20C Flow through of loading ratio of 2:1. FIG. 20D. Purifiedvirus from the loading ratio of 2:1. FIG. 20E Flow through from loadingratio of 3:1. FIG. 20F. Purified virus from the loading ratio of 3:1.(solid line A₂₆₀; dotted line A₂₈₀).

FIG. 21. Isopycnic CsCl ultracentrifugation column purified virus.

FIG. 22. The HPLC profiles of intact viruses present in the columnpurified virus. A. Intact virus B. Defective virus. (solid line A₂₆₀;dotted line A₂₈₀).

FIG. 23. A production and purification flow chart for AdCMVp53

FIG. 24. Kinetics of virus release in the supernatant in a 4×100CellCube™.

FIG. 25. Chromatogram using Source 15Q resin for purification.

FIG. 26. HPLC profile of purified Ad5CMV-p53 product from Source 15Qresin.

FIG. 27. Comparison of bioactivity of original process vs. optimizedprocess to produce Ad5CMV-p53 product.

FIG. 28. Production and Purification flow chart for Ad5CMV-p53 optimizedprocess.

FIG. 29. Lyophilization cycle for adenovirus formulations.

FIG. 30A and FIG. 30B. Storage stability data using secondary drying at10° C. without N₂ blanketing. FIG. 30A, secondary drying at 10° C.without N₂ blanketing for formulation set 10. FIG. 30B, secondary dryingat 10° C. without N₂ blanketing for formulation set 11.

FIG. 31A and FIG. 31B. Storage stability data using secondary drying at30° C. without N₂ blanketing. FIG. 31A, secondary drying at 30° C.without N₂ blanketing for formulation set 10. FIG. 31B, secondary dryingat 30° C. without N₂ blanketing for formulation set 11.

FIG. 32A and FIG. 32B. Storage stability data using secondary drying at30° C. with N₂ blanketing. FIG. 32A, secondary drying at 30° C. with N₂blanketing for formulation set 10. FIG. 32B, secondary drying at 30° C.with N₂ blanketing for formulation set 11.

FIG. 33. Stability data for liquid formulation set #1.

FIG. 34. Stability data for liquid formulation set #2.

FIG. 35. Stability data for liquid formulation set #3.

FIG. 36. Stability data for liquid formulation set #4.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

It has been shown that adenoviral vectors can successfully be used ineukaryotic gene expression and vaccine development. Recently, animalstudies have demonstrated that recombinant adenovirus could be used forgene therapy. Successful studies in administering recombinant adenovirusto different tissues have proven'the effectiveness of adenoviral vectorsin therapy. This success has led to the use of such vectors in humanclinical trials. There now is an increased demand for the production ofadenoviral vectors to be used in various therapies. The techniquescurrently available are insufficient to meet such a demand. The presentinvention provides methods for the production of large amounts ofadenovirus for use in such therapies.

The present invention involves a process that has been developed for theproduction and purification of a replication deficient recombinantadenovirus. The production process is based on the use of a cell culturebioreactor for cell growth and virus production. After viral infectionof the producer cells, virus can be harvested by any number of methods,including virus autolysis or chemical lysis. The harvested crude virussolution can then be purified using a single ion exchange chromatographyrun, after concentration/diafiltration and nuclease treatment to reducethe contaminating nucleic acid concentration in the crude virussolution. The column purified virus has equivalent purity relative tothat of virus purified by cesium banding. The total process recovery ofthe virus product is 70%±10%. This is a significant improvement over theresults reported by Huyghe et al. (1996). Compared to double CsClgradient ultracentrifugation, column purification has the advantage ofbeing more consistent, scaleable, validatable, faster and lessexpensive. This new process represents a significant improvement in thetechnology for manufacturing of adenoviral vectors for gene therapy.

Therefore, the present invention is designed to take advantage of theseimprovements in large scale culturing systems and purification for thepurpose of producing and purifying adenoviral vectors. The variouscomponents for such a system, and methods of producing adenovirustherewith, are set forth in detail below.

1. Host Cells

A) Cells

In a preferred embodiment, the generation and propagation of theadenoviral vectors depend on a unique helper cell line, designated 293,which was transformed from human embryonic kidney cells by Adenovirusserotype 5 (AdS) DNA fragments and constitutively expresses E1 proteins(Graham et al., 1977). Since the E3 region is dispensable from the Adgenome (Jones and Shenk, 1978), the current Ad vectors, with the help of293 cells, carry foreign DNA in either the E1, the E3 or both regions(Graham and Prevec, 1991; Bett et al., 1994).

A first aspect of the present invention is the recombinant cell lineswhich express part of the adenoviral genome. These cells lines arecapable of supporting replication of adenovirus recombinant vectors andhelper viruses having defects in certain adenoviral genes, i.e., are“permissive” for growth of these viruses and vectors. The recombinantcell also is referred to as a helper cell because of the ability tocomplement defects in, and support replication of,replication-incompetent adenoviral vectors. The prototype for anadenoviral helper cell is the 293 cell line, which contains theadenoviral E1 region. 293 cells support the replication of adenoviralvectors lacking E1 functions by providing in trans the E1-activeelements necessary for replication. Other cell lines which also supportthe growth of adenoviruses lacking E1 function include PER.C6(IntroGene, NL), 911 (IntroGene, NL), and IT293SF.

Helper cells according to the present invention are derived from amammalian cell and, preferably, from a primate cell such as humanembryonic kidney cell. Although various primate cells are preferred andhuman or even human embryonic kidney cells are most preferred, any typeof cell that is capable of supporting replication of the virus would beacceptable in the practice of the invention. Other cell types mightinclude, but are not limited to Vero cells, HeLa cells or any eukaryoticcells for which tissue culture techniques are established as long as thecells are adenovirus permissive. The term “adenovirus permissive” meansthat the adenovirus or adenoviral vector is able to complete the entireintracellular virus life cycle within the cellular environment.

The helper cell may be derived from an existing cell line, e.g., from a293 cell line, or developed de novo. Such helper cells express theadenoviral genes necessary to complement in trans deletions in anadenoviral genome or which support replication of an otherwise defectiveadenoviral vector, such as the E1, E2, E4, E5 and late functions. Aparticular portion of the adenovirus genome, the E1 region, has alreadybeen used to generate complementing cell lines. Whether integrated orepisomal, portions of the adenovirus genome lacking a viral origin ofreplication, when introduced into a cell line, will not replicate evenwhen the cell is superinfected with wild-type adenovirus. In addition,because the transcription of the major late unit is after viral DNAreplication, the late functions of adenovirus cannot be expressedsufficiently from a cell line. Thus, the E2 regions, which overlap withlate functions (L1–5), will be provided by helper viruses and not by thecell line. Typically, a cell line according to the present inventionwill express E1 and/or E4.

As used herein, the term “recombinant” cell is intended to refer to acell into which a gene, such as a gene from the adenoviral genome orfrom another cell, has been introduced. Therefore, recombinant cells aredistinguishable from naturally-occurring cells which do not contain arecombinantly-introduced gene. Recombinant cells are thus cells having agene or genes introduced through “the hand of man.”

Replication is determined by contacting a layer of uninfected cells, orcells infected with one or more helper viruses, with virus particles,followed by incubation of the cells. The formation of viral plaques, orcell free areas in the cell layer, is the result of cell lysis caused bythe expression of certain viral products. Cell lysis is indicative ofviral replication.

Examples of other useful mammalian cell lines that may be used with areplication competent virus or converted into complementing host cellsfor use with replication deficient virus are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, HepG2, 3T3, RIN,MDCK and A549 cells.

B) Growth in Selection Media

In certain embodiments, it may be useful to employ selection systemsthat preclude growth of undesirable cells. This may be accomplished byvirtue of permanently transforming a cell line with a selectable markeror by transducing or infecting a cell line with a viral vector thatencodes a selectable marker. In either situation, culture of thetransformed/transduced cell with an appropriate drug or selectivecompound will result in the enhancement, in the cell population, ofthose cells carrying the marker.

Examples of markers include, but are not limited to, HSV thymidinekinase, hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to methotrexate; gpt,that confers resistance to mycophenolic acid; neo, that confersresistance to the aminoglycoside G418; and hygro, that confersresistance to hygromycin.

C. Growth in Serum Weaning

Serum weaning adaptation of anchorage-dependent cells into serum-freesuspension cultures have been used for the production of recombinantproteins (B3erg, 1993) and viral vaccines (Perrin, 1995). There havebeen few reports on the adaptation of 293A cells into serum-freesuspension cultures until recently. Gilbert reported the adaptation of293A cells into serum-free suspension cultures for adenovirus andrecombinant protein production (Gilbert, 1996). A similar adaptationmethod had been used for the adaptation of A549 cells into serum-freesuspension culture for adenovirus production (Morris et al., 1996).Cell-specific virus yields in the adapted suspension cells, however, areabout 5–10-fold lower than those achieved in the parental attachedcells.

Using the similar serum weaning procedure, the inventors havesuccessfully adapted the 293A cells into serum-free suspension culture(293SF cells). In this procedure, the 293 cells were adapted to acommercially available 293 media by sequentially lowering down the FBSconcentration in T-flasks. Briefly, the initial serum concentration inthe media was approximately 10% FBS DMEM media in T-75 flask and thecells were adapted to serum-free IS 293 media in T-flasks by loweringdown the FBS concentration in the media sequentially. After 6 passagesin T-75 flasks the FBS% was estimated to be about 0.019% in the 293cells. The cells were subcultured two more times in the T flasks beforethey were transferred to spinner flasks. The results described hereinbelow show that cells grow satisfactorily in the serum-free medium(IS293 medium, Irvine Scientific, Santa Ana, Calif.). Average doublingtime of the cells were 18–24 h achieving stationary cell concentrationsin the order of 4–10×106 cells/ml without medium exchange.

D. Adaptation of Cells for Suspension Culture

Two methodologies have been used to adapt 293 cells into suspensioncultures. Graham adapted 293A cells into suspension culture (293N3Scells) by 3 serial passages in nude mice (Graham, 1987). The suspension293N3S cells were found to be capable of supporting E1⁻ adenoviralvectors. However, Garnier et al. (1994) observed that the 293N3S cellshad a relatively long initial lag phase in suspension, a low growthrate, and a strong tendency to clump.

The second method that has been used is a gradual adaptation of 293Acells into suspension growth (Cold Spring Harbor Laboratories, 293Scells). Garnier et al. (1994) reported the use of 293S cells forproduction of recombinant proteins from adenoviral vectors. The authorsfound that 293S cells were much less clumpy in calcium-free media and afresh medium exchange at the time of virus infection could significantlyincrease the protein production. It was found that glucose was thelimiting factor in culture without medium exchange.

In the present invention, the 293 cells adapted for growth in serum-freeconditions were adapted into a suspension culture. The cells weretransferred in a serum-free 250 mL spinner suspension culture (100 mLworking volume) for the suspension culture at an initial cell density ofbetween about 1.18E+5 vc/mL and about 5.22E+5 viable cells/mL. The mediamay be supplemented with heparin to prevent aggregation of cells. Thiscell culture systems allows for some increase of cell density whilstcell viability is maintained. Once these cells are growing in culture,the cells are subcultured in the spinner flasks approximately 7 morepassages. It may be noted that the doubling time of the cells isprogressively reduced until at the end of the successive passages thedoubling time is about 1.3 day, i.e. comparable to 1.2 day of the cellsin 10% FBS media in the attached cell culture. In the serum-free IS 293media supplemented with heparin almost all the cells existed asindividual cells not forming aggregates of cells in the suspensionculture.

2. Cell Culture Systems

In any cell culture system, there is a characteristic growth patternfollowing inoculation that includes a lag phase, an accelerated growthphase, an exponential or “log” phase, a negative growth accelerationphase and a plateau or stationary phase. The log and plateau phases givevital information about the cell line, the population doubling timeduring log growth, the growth rate, and the maximum cell densityachieved in plateau. In the log phase, as growth continues, the cellsreach their maximum rate of cell division. Numbers of cells increase inlog relationship to time. During this period of most activemultiplication, the logarithms of the numbers of cells counted at shortintervals, plotted against time, produce a straight line. By making onecount at a specified time and a second count after an interval duringthe log phase of growth and knowing the number of elapsed time units,one can calculate the total number of cell divisions or doublings, andboth the growth rate and generation time. Within a few hours or daysafter the commencement of the log phase, the rate of cell divisionbegins to decline and some of the cells begin to die. This is reflectedon the growth curve by a gradual flattening out of the line. Eventuallythe rate of cells dying is essentially equal to the rate of cellsdividing, and the total viable population remains the same for a periodof time. This is known as the stationary or plateau phase and isrepresented on the growth curve as a flattening out of the line wherethe slope approaches zero.

Measurement of the population doubling time can be used to quantify theresponse of the cells to different inhibitory or stimulatory cultureconditions such as variations in nutrient concentration, hormonaleffects, or toxic drugs. It is also a good monitor of the culture duringserial passage and enables the calculation of cell yields and thedilution factor required at subculture.

The population doubling time is an average figure and describes the netresult of a wide range of cell division rates, including zero, withinthe culture. The doubling time will also differ with varying cell types,culture conditions, and culture vessels. Single time points areunsatisfactory for monitoring growth when the shape of the cell growthcurve is not known. Thus it is important to determine the growth curvefor each cell type being used in the conditions that are being used forthe cell culture. Typical growth curves are sigmoidal in shape, with thefirst part of the curve representing the lag phase, the center part ofthe curve representing the log phase, and the last part of the curverepresenting the plateau phase. The log phase is when the cells aregrowing at the highest rate, and as the cells reach their saturationdensity, their growth will slow and the culture will enter the plateauphase. A detailed description of cell culture techniques and theory canbe found in Freshney, 1992 and Freshney, 1987.

An important aspect of the present invention is infection of theproducer cells with recombinant adenovirus at an appropriate time toachieve maximal virus production. The inventors have found that maximalvirus production is obtained when the producer cells are infectedbetween about when the cells reach the first inflection point on the logphase of the cell growth curve, i.e. mid-log phase, and before the2^(nd) inflection point on the plateau phase of the cell growth curve,i.e. mid-plateau phase. This range can be determined easily for any celltype and any culture conditions with any cell culturing apparatus. Theinflection points on a cell growth curve are when the shape of the linechanges from a convex to a concave shape, or from a concave to a convexshape.

For most growth curves plotted on semi-log scales, the log phase ofgrowth can be approximately represented by a linear increase in theslope of the line over time. That is, at any short interval between twopoints on the line of the logarithmic phase of the curve, the log ofcell number is increasing in a linear fashion relative to time. Thus midlog phase can be approximately defined as the point or interval withinthe log phase in which the cells are dividing at their maximal rate, andthe increase in logs of cell number is linear with respect to time. Latelog phase can be defined as approximately the point or interval of timein which the rate of cell division has slowed, and the log of number ofcells is no longer increasing in a linear fashion with respect to time.When looking at a growth curve, this area would be represented bygradual falling or flattening of the slope of the line. At earlystationary phase, the rate of cell growth is decreasing and gettingnearer the rate of cell death, and thus the slope of the line on thegrowth curve is even less than that at late log phase. At mid-stationaryphase, the rate of cell growth is approximately equal to the rate ofcell division and thus the line on the growth curve is relatively flatand has a slope approaching zero. It will be understood that the skilledartisan can formulate growth curves for any such cell line and identifythe aforementioned regions on the curve.

The ability to produce infectious viral vectors is increasinglyimportant to the pharmaceutical industry, especially in the context ofgene therapy. Over the last decade, advances in biotechnology have ledto the production of a number of important viral vectors that havepotential uses as therapies, vaccines and protein production machines.The use of viral vectors in mammalian cultures has advantages overproteins produced in bacterial or other lower lifeform hosts in theirability to post-translationally process complex protein structures suchas disulfide-dependent folding and glycosylation.

Development of cell culture for production of virus vectors has beengreatly aided by the development in molecular biology of techniques fordesign and construction of vector systems highly efficient in mammaliancell cultures, a battery of useful selection markers, gene amplificationschemes and a more comprehensive understanding of the biochemical andcellular mechanisms involved in procuring the final biologically-activemolecule from the introduced vector.

Frequently, factors which affect the downstream (in this case, beyondthe cell lysis) side of manufacturing scale-up were not consideredbefore selecting the cell line as the host for the expression system.Also, development of bioreactor systems capable of sustaining very highdensity cultures for prolonged periods of time have not lived up to theincreasing demand for increased production at lower costs.

The present invention will take advantage of the recently availablebioreactor technology. Growing cells according to the present inventionin a bioreactor allows for large scale production of fullybiologically-active cells capable of being infected by the adenoviralvectors of the present invention. By operating the system at a lowperfusion rate and applying a different scheme for purification of theinfecting particles, the invention provides a purification strategy thatis easily scaleable to produce large quantities of highly purifiedproduct.

Bioreactors have been widely used for the production of biologicalproducts from both suspension and anchorage dependent animal cellcultures. The most widely used producer cells for adenoviral vectorproduction are anchorage dependent human embryonic kidney cells (293cells). Bioreactors to be developed for adenoviral vector productionshould have the characteristic of high volume-specific culture surfacearea in order to achieve high producer cell density and high virusyield. Microcarrier cell culture in stirred tank bioreactor providesvery high volume-specific culture surface area and has been used for theproduction of viral vaccines (Griffiths, 1986). Furthermore, stirredtank bioreactors have industrially been proven to be scaleable. Themultiplate Cellcube™ cell culture system manufactured by Corning-Costaralso offers a very high volume-specific culture surface area. Cells growon both sides of the culture plates hermetically sealed together in theshape of a compact cube. Unlike stirred tank bioreactors, the Cellcube™culture unit is disposable. This is very desirable at the early stageproduction of clinical product because of the reduced capitalexpenditure, quality control and quality assurance costs associated withdisposable systems. In consideration of the advantages offered by thedifferent systems, both the stirred tank bioreactor and the Cellcube™system were evaluated for the production of adenovirus.

Table 1 list several exemplary techniques for cell culturing and viralparticle production. Currently, there are no methods employed thatresult in both high purity and a high number of viral particles. Thus,the following methods are considered in combination with the large scaleprocess for the production and purification of adenovirus described inthe present invention.

TABLE 1 Virus Particles 5 × 10¹⁴ 1 × 10¹⁵ 1 × 10¹⁶ 1 × 10¹⁷ 1 × 10¹⁸Exemplary Cellcube™ Cellcube™ Packed Bed 1000–5000 L 10,000– Techniquesfor per 10 L Stirred Tank 20,000 L Viral Particle Airlift ReactorStirred Production Tank Total Cell 5 × 10¹⁰ 1 × 10¹¹ 1 × 10¹² 1 × 10¹³ 1× 10¹⁴ Number

A) Anchorage-Dependent Versus Non-Anchorage-Dependent Cultures.

Animal and human cells can be propagated in vitro in two modes: asnon-anchorage dependent cells growing freely in suspension throughoutthe bulk of the culture; or as anchorage-dependent cells requiringattachment to a solid substrate for their propagation (i.e., a monolayertype of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. Large scale suspension culturebased on microbial (bacterial and yeast) fermentation technology hasclear advantages for the manufacturing of mammalian cell products. Theprocesses are relatively simple to operate and straightforward to scaleup. Homogeneous conditions can be provided in the reactor which allowsfor precise monitoring and control of temperature, dissolved oxygen, andpH, and ensures that representative samples of the culture can be taken.

However, suspension cultured cells cannot always be used in theproduction of biologicals. Suspension cultures are still considered tohave tumorigenic potential and thus their use as substrates forproduction put limits on the use of the resulting products in human andveterinary applications (Petricciani, 1985; Larsson, 1987). Virusespropagated in suspension cultures as opposed to anchorage-dependentcultures can sometimes cause rapid changes in viral markers, leading toreduced immunogenicity (Bahnemann, 1980). Finally, sometimes evenrecombinant cell lines can secrete considerably higher amounts ofproducts when propagated as anchorage-dependent cultures as comparedwith the same cell line in suspension (Nilsson and Mosbach, 1987). Forthese reasons, different types of anchorage-dependent cells are usedextensively in the production of different biological products.

B) Reactors and Processes for Suspension.

Large scale suspension culture of mammalian cells in stirred tanks wasundertaken. The instrumentation and controls for bioreactors adapted,along with the design of the fermentors, from related microbialapplications. However, acknowledging the increased demand forcontamination control in the slower growing mammalian cultures, improvedaseptic designs were quickly implemented, improving dependability ofthese reactors. Instrumentation and controls are basically the same asfound in other fermentors and include agitation, temperature, dissolvedoxygen, and pH controls. More advanced probes and autoanalyzers foron-line and off-line measurements of turbidity (a function of particlespresent), capacitance (a function of viable cells present),glucose/lactate, carbonate/bicarbonate and carbon dioxide are available.Maximum cell densities obtainable in suspension cultures are relativelylow at about 2–4×10⁶ cells/ml of medium (which is less than 1 mg drycell weight per ml), well below the numbers achieved in microbialfermentation.

Two suspension culture reactor designs are most widely used in theindustry due to their simplicity and robustness of operation—the stirredreactor and the airlift reactor. The stirred reactor design hassuccessfully been used on a scale of 8000 liter capacity for theproduction of interferon (Phillips et al., 1985; Mizrahi, 1983). Cellsare grown in a stainless steel tank with a height-to-diameter ratio of1:1 to 3:1. The culture is usually mixed with one or more agitators,based on bladed disks or marine propeller patterns. Agitator systemsoffering less shear forces than blades have been described. Agitationmay be driven either directly or indirectly by magnetically coupleddrives. Indirect drives reduce the risk of microbial contaminationthrough seals on stirrer shafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gasses and generatesrelatively low shear forces.

Most large-scale suspension cultures are operated as batch or fed-batchprocesses because they are the most straightforward to operate and scaleup. However, continuous processes based on chemostat or perfusionprinciples are available.

A batch process is a closed system in which a typical growth profile isseen. A lag phase is followed by exponential, stationary and declinephases. In such a system, the environment is continuously changing asnutrients are depleted and metabolites accumulate. This makes analysisof factors influencing cell growth and productivity, and henceoptimization of the process, a complex task. Productivity of a batchprocess may be increased by controlled feeding of key nutrients toprolong the growth cycle. Such a fed-batch process is still a closedsystem because cells, products and waste products are not removed.

In what is still a closed system, perfusion of fresh medium through theculture can be achieved by retaining the cells with a variety of devices(e.g. fine mesh spin filter, hollow fiber or flat plate membranefilters, settling tubes). Spin filter cultures can produce celldensities of approximately 5×10⁷ cells/ml. A true open system and thesimplest perfusion process is the chemostat in which there is an inflowof medium and an outflow of cells and products. Culture medium is fed tothe reactor at a predetermined and constant rate which maintains thedilution rate of the culture at a value less than the maximum specificgrowth rate of the cells (to prevent washout of the cell mass from thereactor). Culture fluid containing cells and cell products andbyproducts is removed at the same rate.

C) Non-Perfused Attachment Systems.

Traditionally, anchorage-dependent cell cultures are propagated on thebottom of small glass or plastic vessels. The restrictedsurface-to-volume ratio offered by classical and traditional techniques,suitable for the laboratory scale, has created a bottleneck in theproduction of cells and cell products on a large scale. In an attempt toprovide systems that offer large accessible surfaces for cell growth insmall culture volume, a number of techniques have been proposed: theroller bottle system, the stack plates propagator, the spiral filmbottles, the hollow fiber system, the packed bed, the plate exchangersystem, and the membrane tubing reel. Since these systems arenon-homogeneous in their nature, and are sometimes based on multipleprocesses, they suffer from the following shortcomings—limited potentialfor scale-up, difficulties in taking cell samples, limited potential formeasuring and controlling key process parameters and difficulty inmaintaining homogeneous environmental conditions throughout the culture.

Despite these drawbacks, a commonly used process for large scaleanchorage-dependent cell production is the roller bottle. Being littlemore than a large, differently shaped T-flask, simplicity of the systemmakes it very dependable and, hence, attractive. Fully automated robotsare available that can handle thousands of roller bottles per day, thuseliminating the risk of contamination and inconsistency associated withthe otherwise required intense human handling. With frequent mediachanges, roller bottle cultures can achieve cell densities of close to0.5×10⁶ cells/cm² (corresponding to approximately 10⁹ cells/bottle oralmost 10⁷ cells/ml of culture media).

D) Cultures on Microcarriers

In an effort to overcome the shortcomings of the traditionalanchorage-dependent culture processes, van Wezel (1967) developed theconcept of the microcarrier culturing systems. In this system, cells arepropagated on the surface of small solid particles suspended in thegrowth medium by slow agitation. Cells attach to the microcarriers andgrow gradually to confluency on the microcarrier surface. In fact, thislarge scale culture system upgrades the attachment dependent culturefrom a single disc process to a unit process in which both monolayer andsuspension culture have been brought together. Thus, combining thenecessary surface for a cell to grow with the advantages of thehomogeneous suspension culture increases production.

The advantages of microcarrier cultures over most otheranchorage-dependent, large-scale cultivation methods are several fold.First, microcarrier cultures offer a high surface-to-volume ratio(variable by changing the carrier concentration) which leads to highcell density yields and a potential for obtaining highly concentratedcell products. Cell yields are up to 1–2×10⁷ cells/ml when cultures arepropagated in a perfused reactor mode. Second, cells can be propagatedin one unit process vessels instead of using many small low-productivityvessels (ie., flasks or dishes). This results in far better nutrientutilization and a considerable saving of culture medium. Moreover,propagation in a single reactor leads to reduction in need for facilityspace and in the number of handling steps required per cell, thusreducing labor cost and risk of contamination. Third, the well-mixed andhomogeneous microcarrier suspension culture makes it possible to monitorand control environmental conditions (e.g., pH, pO₂, and concentrationof medium components), thus leading to more reproducible cellpropagation and product recovery. Fourth, it is possible to take arepresentative sample for microscopic observation, chemical testing, orenumeration. Fifth, since microcarriers settle out of suspensionquickly, use of a fed-batch process or harvesting of cells can be donerelatively easily. Sixth, the mode of the anchorage-dependent culturepropagation on the microcarriers makes it possible to use this systemfor other cellular manipulations, such as cell transfer without the useof proteolytic enzymes, cocultivation of cells, transplantation intoanimals, and perfusion of the culture using decanters, columns,fluidized beds, or hollow fibers for microcarrier retainment. Seventh,microcarrier cultures are relatively easily scaled up using conventionalequipment used for cultivation of microbial and animal cells insuspension.

E) Microencapsulation of Mammalian Cells

One method which has shown to be particularly useful for culturingmammalian cells is microencapsulation. The mammalian cells are retainedinside a semipermeable hydrogel membrane. A porous membrane is formedaround the cells permitting the exchange of nutrients, gases, andmetabolic products with the bulk medium surrounding the capsule. Severalmethods have been developed that are gentle, rapid and non-toxic andwhere the resulting membrane is sufficiently porous and strong tosustain the growing cell mass throughout the term of the culture. Thesemethods are all based on soluble alginate gelled by droplet contact witha calcium-containing solution. Lim (1982, U.S. Pat. No. 4,352,883,incorporated herein by reference,) describes cells concentrated in anapproximately 1% solution of sodium alginate which are forced through asmall orifice, forming droplets, and breaking free into an approximately1% calcium chloride solution. The droplets are then cast in a layer ofpolyamino acid that ionically bonds to the surface alginate. Finally thealginate is reliquefied by treating the droplet in a chelating agent toremove the calcium ions. Other methods use cells in a calcium solutionto be dropped into a alginate solution, thus creating a hollow alginatesphere. A similar approach involves cells in a chitosan solution droppedinto alginate, also creating hollow spheres.

Microencapsulated cells are easily propagated in stirred tank reactorsand, with beads sizes in the range of 150–1500 μm in diameter, areeasily retained in a perfused reactor using a fine-meshed screen. Theratio of capsule volume to total media volume can be maintained from asdense as 1:2 to 1:10. With intracapsular cell densities of up to 10⁸,the effective cell density in the culture is 1–5×10⁷.

The advantages of microencapsulation over other processes include theprotection from the deleterious effects of shear stresses which occurfrom sparging and agitation, the ability to easily retain beads for thepurpose of using perfused systems, scale up is relativelystraightforward and the ability to use the beads for implantation.

The current invention includes cells which are anchorage-dependent innature. 293 cells, for example, are anchorage-dependent, and when grownin suspension, the cells will attach to each other and grow in clumps,eventually suffocating cells in the inner core of each clump as theyreach a size that leaves the core cells unsustainable by the cultureconditions. Therefore, an efficient means of large-scale culture ofanchorage-dependent cells is needed in order to effectively employ thesecells to generate large quantities of adenovirus.

F) Perfused Attachment Systems

Perfused attachment systems are a preferred form of the presentinvention. Perfusion refers to continuous flow at a steady rate, throughor over a population of cells (of a physiological nutrient solution). Itimplies the retention of the cells within the culture unit as opposed tocontinuous-flow culture which washes the cells out with the withdrawnmedia (e.g., chemostat). The idea of perfusion has been known since thebeginning of the century, and has been applied to keep small pieces oftissue viable for extended microscopic observation. The technique wasinitiated to mimic the cells milieu in vivo where cells are continuouslysupplied with blood, lymph, or other body fluids. Without perfusion,cells in culture go through alternating phases of being fed and starved,thus limiting full expression of their growth and metabolic potential.

The current use of perfused culture is in response to the challenge ofgrowing cells at high densities (i.e., 0.1–5×10⁸ cells/ml). In order toincrease densities beyond 2–4×10⁶ cells/ml, the medium has to beconstantly replaced with a fresh supply in order to make up fornutritional deficiencies and to remove toxic products. Perfusion allowsfor a far better control of the culture environment (pH, pO₂, nutrientlevels, etc.) and is a means of significantly increasing the utilizationof the surface area within a culture for cell attachment.

The development of a perfused packed-bed reactor using a bed matrix of anon-woven fabric has provided a means for maintaining a perfusionculture at densities exceeding 10⁸ cells/ml of the bed volume(CelliGen™, New Brunswick Scientific, Edison, N.J.; Wang et al., 1992;Wang et al., 1993; Wang et al., 1994). Briefly described, this reactorcomprises an improved reactor for culturing of both anchorage- andnon-anchorage-dependent cells. The reactor is designed as a packed bedwith a means to provide internal recirculation. Preferably, a fibermatrix carrier is placed in a basket within the reactor vessel. A topand bottom portion of the basket has holes, allowing the medium to flowthrough the basket. A specially designed impeller provides recirculationof the medium through the space occupied by the fiber matrix forassuring a uniform supply of nutrient and the removal of wastes. Thissimultaneously assures that a negligible amount of the total cell massis suspended in the medium. The combination of the basket and therecirculation also provides a bubble-free flow of oxygenated mediumthrough the fiber matrix. The fiber matrix is a non-woven fabric havinga “pore” diameter of from 10 μm to 100 μm, providing for a high internalvolume with pore volumes corresponding to 1 to 20 times the volumes ofindividual cells.

In comparison to other culturing systems, this approach offers severalsignificant advantages. With a fiber matrix carrier, the cells areprotected against mechanical stress from agitation and foaming. The freemedium flow through the basket provides the cells with optimum regulatedlevels of oxygen, pH, and nutrients. Products can be continuouslyremoved from the culture and the harvested products are free of cellsand can be produced in low-protein medium which facilitates subsequentpurification steps. Also, the unique design of this reactor systemoffers an easier way to scale up the reactor. Currently, sizes up to 30liter are available. One hundred liter and 300 liter versions are indevelopment and theoretical calculations support up to a 1000 literreactor. This technology is explained in detail in WO 94/17178 (Aug. 4,1994, Freedman et al.), which is hereby incorporated by reference in itsentirety.

The Cellcube™ (Corning-Costar) module provides a large styrenic surfacearea for the immobilization and growth of substrate attached cells. Itis an integrally encapsulated sterile single-use device that has aseries of parallel culture plate joined to create thin sealed laminarflow spaces between adjacent plates.

The Cellcube™ module has inlet and outlet ports that are diagonallyopposite each other and help regulate the flow of media. During thefirst few days of growth the culture is generally satisfied by the mediacontained within the system after initial seeding. The amount of timebetween the initial seeding and the start of the media perfusion isdependent on the density of cells in the seeding inoculum and the cellgrowth rate. The measurement of nutrient concentration in thecirculating media is a good indicator of the status of the culture. Whenestablishing a procedure it may be necessary to monitor the nutrientscomposition at a variety of different perfusion rates to determine themost economical and productive operating parameters.

Cells within the system reach a higher density of solution (cells/ml)than in traditional culture systems. Many typically used basal media aredesigned to support 1–2×10⁶ cells/ml/day. A typical Cellcube™, run withan 85,000 cm surface, contains approximately 6L media within the module.The cell density often exceeds 10⁷ cells/mL in the culture vessel. Atconfluence, 2–4 reactor volumes of media are required per day.

The timing and parameters of the production phase of cultures depends onthe type and use of a particular cell line. Many cultures require adifferent media for production than is required for the growth phase ofthe culture. The transition from one phase to the other will likelyrequire multiple washing steps in traditional cultures. However, theCellcube™ system employs a perfusion system. On of the benefits of sucha system is the ability to provide a gentle transition between variousoperating phases. The perfusion system negates the need for traditionalwash steps that seek to remove serum components in a growth medium.

In an exemplary embodiment of the present invention, the CellCube™system is used to grow cells transfected with AdCMVp53. 293 cells wereinoculated into the Cellcube™ according to the manufacturer'srecommendation. Inoculation cell densities were in the range of1–1.5×10⁴/cm². Cells were allowed to grow for 7 days at 37° C. underculture conditions of pH=7.20, DO=60% air saturation. The mediumperfusion rate was regulated according to the glucose concentration inthe Cellcube™. One day before viral infection, medium for perfusion waschanged from a buffer comprising 10% FBS to a buffer comprising 0% FBS.On day 8, cells were infected with virus at a multiplicity of infection(MOI) of 5. Medium perfusion was stopped for 1 hr immediately afterinfection then resumed for the remaining period of the virus productionphase. Culture was harvested 45–48 hr post-infection. Of course theseculture conditions are exemplary and may be varied according to thenutritional needs and growth requirements of a particular cell line.Such variation may be performed without undue experimentation and arewell within the skill of the ordinary person in the art.

G) Serum-Free Suspension Culture

In particular embodiments, adenoviral vectors for gene therapy areproduced from anchorage-dependent culture of 293 cells (293A cells) asdescribed above. Scale-up of adenoviral vector production is constrainedby the anchorage-dependency of 293A cells. To facilitate scale-up andmeet future demand for adenoviral vectors, significant efforts have beendevoted to the development of alternative production processes that areamenable to scale-up. Methods include growing 293A cells in microcarriercultures and adaptation of 293A producer cells into suspension cultures.Microcarrier culture techniques have been described above. Thistechnique relies on the attachment of producer cells onto the surfacesof microcarriers which are suspended in culture media by mechanicalagitation. The requirement of cell attachment may present somelimitations to the scaleability of microcarrier cultures.

Until the present application there have been no reports on the use of293 suspension cells for adenoviral vector production for gene therapy.Furthermore, the reported suspension 293 cells require the presence of5–10% FBS in the culture media for optimal cell growth and virusproduction. Historically, presence of bovine source proteins in cellculture media has been a regulatory concerns, especially recentlybecause of the outbreak of Bovine Spongiform Encephalopathy (BSE) insome countries. Rigorous and complex downstream purification process hasto be developed to remove contaminating proteins and any adventitiousviruses from the final product. Development of serum-free 293 suspensionculture is deemed to be a major process improvement for the productionof adenoviral vector for gene therapy.

Results of virus production in spinner flasks and a 3 L stirred tankbioreactor indicate that cell specific virus productivity of the 293SFcells was approximately 2.5×10⁴ vp/cell, which is approximately 60–90%of that from the 293A cells. However, because of the higher stationarycell concentration, volumetric virus productivity from the 293SF cultureis essentially equivalent to that of the 293A cell culture. Theinventors also observed that virus production increased significantly bycarrying out a fresh medium exchange at the time of virus infection. Theinventors are going to evaluate the limiting factors in the medium.These findings allow for a scaleable, efficient, and easily validatableprocess for the production of adenoviral vector. This adaptation methodis not limited to 293A cells only and will be equally useful whenapplied to other adenoviral vector producer cells.

3. Methods of Cell Harvest and Lysis

Adenoviral infection results in the lysis of the cells being infected.The lytic characteristics of adenovirus infection permit two differentmodes of virus production. One is harvesting infected cells prior tocell lysis. The other mode is harvesting virus supernatant aftercomplete cell lysis by the produced virus. For the latter mode, longerincubation times are required in order to achieve complete cell lysis.This prolonged incubation time after virus infection creates a seriousconcern about increased possibility of generation of replicationcompetent adenovirus (RCA), particularly for the current firstgeneration adenoviral vectors (E1-deleted vector). Therefore, harvestinginfected cells before cell lysis was chosen as the production mode ofchoice. Table 2 lists the most common methods that have been used forlysing cells after cell harvest.

TABLE 2 Methods used for cell lysis Methods Procedures CommentsFreeze-thaw Cycling between dry Easy to carry out at lab ice and 37° C.water scale. High cell bath lysis efficiency Not scaleable Notrecommended for large scale manufacturing Solid Shear French PressCapital equipment Hughes Press investment Virus containment concernsLack of experience Detergent lysis Non-ionic detergent Easy to carry outat both solutions such as lab and manufacturing Tween, Triton, NP-40,scale etc. Wide variety of detergent choices Concerns of residualdetergent in finished product Hypotonic solution water, citric bufferLow lysis efficiency lysis Liquid Shear Homogenizer Capital equipmentImpinging Jet investment Microfluidizer Virus containment concernsScaleability concerns Sonication Ultrasound Capital equipment investmentVirus containment concerns Noise pollution Scaleability concern

A) Detergents

Cells are bounded by membranes. In order to release components of thecell, it is necessary to break open the cells. The most advantageous wayin which this can be accomplished, according to the present invention,is to solubilize the membranes with the use of detergents. Detergentsare amphipathic molecules with an apolar end of aliphatic or aromaticnature and a polar end which may be charged or uncharged. Detergents aremore hydrophilic than lipids and thus have greater water solubility thanlipids. They allow for the dispersion of water insoluble compounds intoaqueous media and are used to isolate and purify proteins in a nativeform.

Detergents can be denaturing or non-denaturing. The former can beanionic such as sodium dodecyl sulfate or cationic such as ethyltrimethyl ammonium bromide. These detergents totally disrupt membranesand denature the protein by breaking protein-protein interactions. Nondenaturing detergents can be divided into non-anionic detergents such asTriton®X-100, bile salts such as cholates and zwitterionic detergentssuch as CHAPS. Zwitterionics contain both cationic and anion groups inthe same molecule, the positive electric charge is neutralized by thenegative charge on the same or adjacent molecule.

Denaturing agents such as SDS bind to proteins as monomers and thereaction is equilibrium driven until saturated. Thus, the freeconcentration of monomers determines the necessary detergentconcentration. SDS binding is cooperative i.e. the binding of onemolecule of SDS increase the probability of another molecule binding tothat protein, and alters proteins into rods whose length is proportionalto their molecular weight.

Non-denaturing agents such as Triton®X-100 do not bind to nativeconformations nor do they have a cooperative binding mechanism. Thesedetergents have rigid and bulky apolar moieties that do not penetrateinto water soluble proteins. They bind to the hydrophobic parts ofproteins. Triton®X100 and other polyoxyethylene nonanionic detergentsare inefficient in breaking protein-protein interaction and can causeartifactual aggregations of protein. These detergents will, however,disrupt protein-lipid interactions but are much gentler and capable ofmaintaining the native form and functional capabilities of the proteins.

Detergent removal can be attempted in a number of ways. Dialysis workswell with detergents that exist as monomers. Dialysis is somewhatineffective with detergents that readily aggregate to form micellesbecause the micelles are too large to pass through dialysis. Ionexchange chromatography can be utilized to circumvent this problem. Thedisrupted protein solution is applied to an ion exchange chromatographycolumn and the column is then washed with buffer minus detergent. Thedetergent will be removed as a result of the equilibration of the bufferwith the detergent solution. Alternatively the protein solution may bepassed through a density gradient. As the protein sediments through thegradients the detergent will come off due to the chemical potential.

Often a single detergent is not versatile enough for the solubilizationand analysis of the milieu of proteins found in a cell. The proteins canbe solubilized in one detergent and then placed in another suitabledetergent for protein analysis. The protein detergent micelles formed inthe first step should separate from pure detergent micelles. When theseare added to an excess of the detergent for analysis, the protein isfound in micelles with both detergents. Separation of thedetergent-protein micelles can be accomplished with ion exchange or gelfiltration chromatography, dialysis or buoyant density type separations.

Triton®X-Detergents: This family of detergents (Triton®X-100, X114 andNP-40) have the same basic characteristics but are different in theirspecific hydrophobic-hydrophilic nature. All of these heterogeneousdetergents have a branched 8-carbon chain attached to an aromatic ring.This portion of the molecule contributes most of the hydrophobic natureof the detergent. Triton®X detergents are used to solublize membraneproteins under non-denaturing conditions. The choice of detergent tosolubilize proteins will depend on the hydrophobic nature of the proteinto be solubilized. Hydrophobic proteins require hydrophobic detergentsto effectively solubilize them.

Triton® X-100 and NP40 are very similar in structure and hydrophobicityand are interchangeable in most applications including cell lysis,delipidation protein dissociation and membrane protein and lipidsolubilization. Generally 2 mg detergent is used to solubilize lmgmembrane protein or 10 mg detergent/lmg of lipid membrane. Triton® X-114is useful for separating hydrophobic from hydrophilic proteins.

Brij® Detergents: These are similar in structure to Triton® X detergentsin that they have varying lengths of polyoxyethylene chains attached toa hydrophobic chain. However, unlike Triton® X detergents, the Brij®detergents do not have an aromatic ring and the length of the carbonchains can vary. The Brij detergents are difficult to remove fromsolution using dialysis but may be removed by detergent removing gels.Brij®58 is most similar to Triton®X100 in its hydrophobic/hydrophiliccharacteristics. Brij®-35 is a commonly used detergent in HPLCapplications.

Dializable Nonionic Detergents: η-Octyl-β-D-glucoside(octylglucopyranoside) and η-Octyl-β-D-thioglucoside(octylthioglucopyranoside, OTG) are nondenaturing nonionic detergentswhich are easily dialyzed from solution. These detergents are useful forsolubilizing membrane proteins and have low UV absorbances at 280 nm.Octylglucoside has a high CMC of 23–25 mM and has been used atconcentrations of 1.1–1.2% to solubilize membrane proteins.

Octylthioglucoside was first synthesized to offer an alternative tooctylglucoside. Octylglucoside is expensive to manufacture and there aresome inherent problems in biological systems because it can behydrolyzed by β-glucosidase.

Tween® Detergents: The Tween® detergents are nondenaturing, nonionicdetergents. They are polyoxyethylene sorbitan esters of fatty acids.Tween® 20 and Tween® 80 detergents are used as blocking agents inbiochemical applications and are usually added to protein solutions toprevent nonspecific binding to hydrophobic materials such as plastics ornitrocellulose. They have been used as blocking agents in ELISA andblotting applications. Generally, these detergents are used atconcentrations of 0.01–1.0% to prevent nonspecific binding tohydrophobic materials.

Tween® 20 and other nonionic detergents have been shown to remove someproteins from the surface of nitrocellulose. Tween® 80 has been used tosolubilize membrane proteins, present nonspecific binding of protein tomultiwell plastic tissue culture plates and to reduce nonspecificbinding by serum proteins and biotinylated protein A to polystyreneplates in ELISA.

The difference between these detergents is the length of the fatty acidchain. Tween® 80 is derived from oleic acid with a C₁₈ chain whileTween® 20 is derived from lauric acid with a C₁₂ chain. The longer fattyacid chain makes the Tween® 80 detergent less hydrophilic than Tween® 20detergent. Both detergents are very soluble in water.

The Tween® detergents are difficult to remove from solution by dialysis,but Tween® 20 can be removed by detergent removing gels. Thepolyoxyethylene chain found in these detergents makes them subject tooxidation (peroxide formation) as is true with the Triton® X and Brij®series detergents.

Zwitterionic Detergents: The zwitterionic detergent, CHAPS, is asulfobetaine derivative of cholic acid. This zwitterionic detergent isuseful for membrane protein solubilization when protein activity isimportant. This detergent is useful over a wide range of pH (pH 2–12)and is easily removed from solution by dialysis due to high CMCs (8–10mM). This detergent has low absorbances at 280 nm making it useful whenprotein monitoring at this wavelength is necessary. CHAPS is compatiblewith the BCA Protein Assay and can be removed from solution by detergentremoving gel. Proteins can be iodinated in the presence of CHAPS.

CHAPS has been successfully used to solubilize intrinsic membraneproteins and receptors and maintain the functional capability of theprotein. When cytochrome P-450 is solubilized in either Triton® X-100 orsodium cholate aggregates are formed.

B) Non-Detergent Methods

Various non-detergent methods, though not preferred, may be employed inconjunction with other advantageous aspects of the present invention:

Freeze-Thaw: This has been a widely used technique for lysis cells in agentle and effective manner. Cells are generally frozen rapidly in, forexample, a dry ice/ethanol bath until completely frozen, thentransferred to a 37° C. bath until completely thawed. This cycle isrepeated a number of times to achieve complete cell lysis.

Sonication: High frequency ultrasonic oscillations have been found to beuseful for cell disruption. The method by which ultrasonic waves breakcells is not fully understood but it is known that high transientpressures are produced when suspensions are subjected to ultrasonicvibration. The main disadvantage with this technique is thatconsiderable amounts of heat are generated. In order to minimize heateffects specifically designed glass vessels are used to hold the cellsuspension. Such designs allow the suspension to circulate away from theultrasonic probe to the outside of the vessel where it is cooled as theflask is suspended in ice.

High Pressure Extrusion: This is a frequently used method to disruptmicrobial cell. The French pressure cell employs pressures of 10.4×10⁷Pa (16, 000 p.s.i) to break cells open. These apparatus consists of astainless steel chamber which opens to the outside by means of a needlevalve. The cell suspension is placed in the chamber with the needlevalve in the closed position. After inverting the chamber, the valve isopened and the piston pushed in to force out any air in the chamber.With the valve in the closed position, the chamber is restored to itsoriginal position, placed on a solid based and the required pressure isexerted on the piston by a hydraulic press. When the pressure has beenattained the needle valve is opened fractionally to slightly release thepressure and as the cells expand they burst. The valve is kept openwhile the pressure is maintained so that there is a trickle of rupturedcell which may be collected.

Solid Shear Methods: Mechanical shearing with abrasives may be achievedin Mickle shakers which oscillate suspension vigorously (300–3000time/min) in the presence of glass beads of 500 nm diameter. This methodmay result in organelle damage. A more controlled method is to use aHughes press where a piston forces most cells together with abrasives ordeep frozen paste of cells through a 0.25 mm diameter slot in thepressure chamber. Pressures of up to 5.5×10⁷ Pa (8000 p.s.i.) may beused to lyse bacterial preparations.

Liquid Shear Methods: These methods employ blenders, which use highspeed reciprocating or rotating blades, homogenizers which use anupward/downward motion of a plunger and ball and microfluidizers orimpinging jets which use high velocity passage through small diametertubes or high velocity impingement of two fluid streams. The blades ofblenders are inclined at different angles to permit efficient mixing.Homogenizers are usually operated in short high speed bursts of a fewseconds to minimize local heat. These techniques are not generallysuitable for microbial cells but even very gentle liquid shear isusually adequate to disrupt animal cells.

Hypotonic/Hypertonic Methods: Cells are exposed to a solution with amuch lower (hypotonic) or higher (hypertonic) solute concentration. Thedifference in solute concentration creates an osmotic pressure gradient.The resulting flow of water into the cell in a hypotonic environmentcauses the cells to swell and burst. The flow of water out of the cellin a hypertonic environment causes the cells to shrink and subsequentlyburst.

Viral Lysis Methods: In some situations, the method of viral lysis maybe advantageous to use, and with modifications to the experimentalprotocol, the formation of RCA may be minimized. Since adenoviruses arelytic viruses, after infection of the host cells the mature viruses lysethe cell and are released into the supernatant and then can be harvestedby conventional methods. One of the advantages to using the viral lysismethod is the generation of more mature viral particles, since earlylysis by mechanical or chemical means may lead to increased numbers ofdefective particles. In addition, the process permits an easier and moreprecise follow-up of the production kinetics directly on the homogeneoussamples of supernatant, which produces better reproducibility of theproduction runs. Chemical lysis also presents an additional step in theprocess and requires the removal of the lysis agent, both of which maylead to potential losses of product and/or diminished activity.

In utilizing the viral lysis method, the kinetics of the liberation ofvirions can be followed in different ways and will be able to indicatethe optimal time for supernatant harvest. For example, HPLC, IEC, PCR,dye exclusion, spectrophotometry, ELISA, RIA or nephelometric methodsmay be used. Harvesting is preferably performed when approximatley 50%of the virions have been released. More preferably, the supernatant isharvested when at least 70% of the virions are released, and mostpreferably, the supernatant is harvested when at least 90% of thevirions are released, or when the viral release reaches a plateau asmeasured by one of the methods indicated above. Variations in the timeneeded for the virus release to reach a plateau may be observed whenusing modification of gene transfer vector, however the harvest schedulecan easily be modified by the skilled artisan when using one or more ofthe methods above to follow the kinetics of virus release.

4. Methods of Concentration and Filtration

One aspect of the present invention employs methods of crudepurification of adenovirus from a cell lysate. These methods includeclarification, concentration and diafiltration. The initial step in thispurification process is clarification of the cell lysate to remove largeparticulate matter, particularly cellular components, from the celllysate. Clarification of the lysate can be achieved using a depth filteror by tangential flow filtration. In a preferred embodiment of thepresent invention, the cell lysate is passed through a depth filter,which consists of a packed column of relatively non-adsorbent material(e.g. polyester resins, sand, diatomeceous earth, colloids, gels, andthe like). In tangential flow filtration (TFF), the lysate solutionflows across a Membrane surface which facilitates back diffusion ofsolute from the membrane surface into the bulk solution. Membranes aregenerally arranged within various types of filter apparatus includingopen channel plate and frame, hollow fibers, and tubules.

After clarification and prefiltration of the cell lysate, the resultantvirus supernatant is first concentrated and then the buffer is exchangedby diafiltration. The virus supernatant is concentrated by tangentialflow filtration across an ultrafiltration membrane of 100–300K nominalmolecular weight cutoff. Ultrafiltration is a pressure-modifiedconvective process that uses semi-permeable membranes to separatespecies by molecular size, shape and/or charge. It separates solventsfrom solutes of various sizes, independent of solute molecular size.Ultrafiltration is gentle, efficient and can be used to simultaneouslyconcentrate and desalt solutions. Ultrafiltration membranes generallyhave two distinct layers: a thin (0.1–1.5 μm), dense skin with a porediameter of 10–400 angstroms and an open substructure of progressivelylarger voids which are largely open to the permeate side of theultrafilter. Any species capable of passing through the pores of theskin can therefore freely pass through the membrane. For maximumretention of solute, a membrane is selected that has a nominal molecularweight cut-off well below that of the species being retained. Inmacromolecular concentration, the membrane enriches the content of thedesired biological species and provides filtrate cleared of retainedsubstances. Microsolutes are removed convectively with the solvent. Asconcentration of the retained solute increases, the ultrafiltration ratediminishes.

Diafiltration, or buffer exchange, using ultrafilters is an ideal wayfor removal and exchange of salts, sugars, non-aqueous solventsseparation of free from bound species, removal of material of lowmolecular weight, or rapid change of ionic and pH environments.Microsolutes are removed most efficiently by adding solvent to thesolution being ultrafiltered at a rate equal to the ultrafiltrationrate. This washes microspecies from the solution at constant volume,purifying the retained species. The present invention utilizes adiafiltration step to exchange the buffer of the virus supernatant priorto Benzonase® treatment.

5. Viral Infection

The present invention employs, in one example, adenoviral infection ofcells in order to generate therapeutically significant vectors.Typically, the virus will simply be exposed to the appropriate host cellunder physiologic conditions, permitting uptake of the virus. Thoughadenovirus is exemplified, the present methods may be advantageouslyemployed with other viral vectors, as discussed below.

A) Adenovirus

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized DNA genome, ease of manipulation, high titer,wide target-cell range, and high infectivity. The roughly 36 kB viralgenome is bounded by 100–200 base pair (bp) inverted terminal repeats(ITR), in which are contained cis-acting elements necessary for viralDNA replication and packaging. The early (E) and late (L) regions of thegenome that contain different transcription units are divided by theonset of viral DNA replication.

The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff (Renan, 1990). The products of the late genes (L1, L2, L3, L4 andL5), including the majority of the viral capsid proteins, are expressedonly after significant processing of a single primary transcript issuedby the major late promoter (MLP). The MLP (located at 16.8 map units) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence which makes them preferred mRNAs for translation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.Elimination of large potions of the adenoviral genome, and providing thedelete gene products in trans, by helper virus and/or helper cells,allows for the insertion of large portions of heterologous DNA into thevector. This strategy also will result in reduced toxicity andimmunogenicity of the adenovirus gene products.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100–200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus (Hay et al., 1984). Therefore, inclusion ofthese elements in an adenoviral vector should permit replication.

In addition, the packaging signal for viral encapsidation is localizedbetween 194–385 bp (0.5–1.1 map units) at the left end of the viralgenome (Hearing et al., 1987). This signal mimics the proteinrecognition site in bacteriophage λ DNA where a specific sequence closeto the left end, but outside the cohesive end sequence, mediates thebinding to proteins that are required for insertion of the DNA into thehead structure. E1 substitution vectors of Ad have demonstrated that a450 bp (0–1.25 map units) fragment at the left end of the viral genomecould direct packaging in 293 cells (Levrero et al., 1991).

Previously, it has been shown that certain regions of the adenoviralgenome can be incorporated into the genome of mammalian cells and thegenes encoded thereby expressed. These cell lines are capable ofsupporting the replication of an adenoviral vector that is deficient inthe adenoviral function encoded by the cell line. There also have beenreports of complementation of replication deficient adenoviral vectorsby “helping” vectors, e.g., wild-type virus or conditionally defectivemutants.

Replication-deficient adenoviral vectors can be complemented, in trans,by helper virus. This observation alone does not permit isolation of thereplication-deficient vectors, however, since the presence of helpervirus, needed to provide replicative fimctions, would contaminate anypreparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element, as provided for in thepresent invention, derives from the packaging function of adenovirus.

It has been shown that a packaging signal for adenovirus exists in theleft end of the conventional adenovirus map (Tibbetts, 1977). Laterstudies showed that a mutant with a deletion in the E1A (194–358 bp)region of the genome grew poorly even in a cell line that complementedthe early (E1A) function (Hearing and Shenk, 1983). When a compensatingadenoviral DNA (0–353 bp) was recombined into the right end of themutant, the virus was packaged normally. Further mutational analysisidentified a short, repeated, position-dependent element in the left endof the Ad5 genome. One copy of the repeat was found to be sufficient forefficient packaging if present at either end of the genome, but not whenmoved towards the interior of the Ad5 DNA molecule (Hearing et al.,1987).

By using mutated versions of the packaging signal, it is possible tocreate helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals arepackaged selectively when compared to the helpers. If the preference isgreat enough, stocks approaching homogeneity should be achieved.

B) Retrovirus

Although adenoviral infection of cells for the generation oftherapeutically significant vectors is a preferred embodiments of thepresent invention, it is contemplated that the present invention mayemploy retroviral infection of cells for the purposes of generating suchvectors. The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains threegenes—gag, pol and env—that code for capsid proteins, polymerase enzyme,and envelope components, respectively. A sequence found upstream fromthe gag gene, termed Y, functions as a signal for packaging of thegenome into virions. Two long terminal repeat (LTR) sequences arepresent at the 5′ and 3′ ends of the viral genome. These contain strongpromoter and enhancer sequences and are also required for integration inthe host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding apromoter is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol and envgenes but without the LTR and Y components is constructed (Mann et al.,1983). When a recombinant plasmid containing a human cDNA, together withthe retroviral LTR and Y sequences is introduced into this cell line (bycalcium phosphate precipitation for example), the Y sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of galactose residues to the viralenvelope. This modification could permit the specific infection of cellssuch as hepatocytes via asialoglycoprotein receptors, should this bedesired.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, the infection of a variety of human cellsthat bore those surface antigens was demonstrated with an ecotropicvirus in vitro (Roux et al., 1989).

C) Adeno Associated Virus

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.Inverted terminal repeats flank the genome. Two genes are present withinthe genome, giving rise to a number of distinct gene products. Thefirst, the cap gene, produces three different virion proteins (VP),designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes fournon-structural proteins (NS). One or more of these rep gene products isresponsible for transactivating AAV transcription.

The three promoters in AAV are designated by their location, in mapunits, in the genome. These are, from left to right, p5, p19 and p40.Transcription gives rise to six transcripts, two initiated at each ofthree promoters, with one of each pair being spliced. The splice site,derived from map units 42–46, is the same for each transcript. The fournon-structural proteins apparently are derived from the longer of thetranscripts, and three virion proteins all arise from the smallesttranscript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

The terminal repeats of the AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as p201, which containsa modified AAV genome (Samulski et al. 1987), or by other methods knownto the skilled artisan, including but not limited to chemical orenzymatic synthesis of the terminal repeats based upon the publishedsequence of AAV. The ordinarily skilled artisan can determine, bywell-known methods such as deletion analysis, the minimum sequence orpart of the AAV ITRs which is required to allow function, i.e., stableand site-specific integration. The ordinarily skilled artisan also candetermine which minor modifications of the sequence can be toleratedwhile maintaining the ability of the terminal repeats to direct stable,site-specific integration.

AAV-based vectors have proven to be safe and effective vehicles for genedelivery in vitro, and these vectors are being developed and tested inpre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo (Carter and Flotte,1996;

Chatterjee et al., 1995; Ferrari et al., 1996; Fisher et al., 1996;Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994; 1996,Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al., 1996; Xiaoet al., 1996).

AAV-mediated efficient gene transfer and expression in the lung has ledto clinical trials for the treatment of cystic fibrosis (Carter andFlotte, 1996; Flotte et al., 1993). Similarly, the prospects fortreatment of muscular dystrophy by AAV-mediated gene delivery of thedystrophin gene to skeletal muscle, of Parkinson's disease by tyrosinehydroxylase gene delivery to the brain, of hemophilia B by Factor IXgene delivery to the liver, and potentially of myocardial infarction byvascular endothelial growth factor gene to the heart, appear promisingsince AAV-mediated transgene expression in these organs has recentlybeen shown to be highly efficient (Fisher et al., 1996; Flotte et al.,1993; Kaplitt et al., 1994;

D) Herpesvirus

Because herpes simplex virus (HSV) is neurotropic, it has generatedconsiderable interest in treating nervous system disorders. Moreover,the ability of HSV to establish latent infections in non-dividingneuronal cells without integrating in to the host cell chromosome orotherwise altering the host cell's metabolism, along with the existenceof a promoter that is active during latency makes HSV an attractivevector. And though much attention has focused on the neurotropicapplications of HSV, this vector also can be exploited for other tissuesgiven its wide host range.

Another factor that makes HSV an attractive vector is the size andorganization of the genome. Because HSV is large, incorporation ofmultiple genes or expression cassettes is less problematic than in othersmaller viral systems. In addition, the availability of different viralcontrol sequences with varying performance (temporal, strength, etc.)makes it possible to control expression to a greater extent than inother systems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations.

HSV also is relatively easy to manipulate and can be grown to hightiters. Thus, delivery is less of a problem, both in terms of volumesneeded to attain sufficient MOI and in a lessened need for repeatdosings. For a review of HSV as a gene therapy vector, see Glorioso etal. (1995).

HSV, designated with subtypes 1 and 2, are enveloped viruses that areamong the most common infectious agents encountered by humans, infectingmillions of human subjects worldwide. The large, complex,double-stranded DNA genome encodes for dozens of different geneproducts, some of which derive from spliced transcripts. In addition tovirion and envelope structural components, the virus encodes numerousother proteins including a protease, a ribonucleotides reductase, a DNApolymerase, a ssDNA binding protein, a helicase/primase, a DNA dependentATPase, a dUTPase and others.

HSV genes form several groups whose expression is coordinately regulatedand sequentially ordered in a cascade fashion (Honess and Roizman, 1974;Honess and Roizman 1975; Roizman and Sears, 1995). The expression of αgenes, the first set of genes to be expressed after infection, isenhanced by the virion protein number 16, or α-transinducing factor(Post et al., 1981; Batterson and Roizman, 1983; Campbell, et al.,1983). The expression of β genes requires functional α gene products,most notably ICP4, which is encoded by the α4 gene (DeLuca et al.,1985). γ genes, a heterogeneous group of genes encoding largely virionstructural proteins, require the onset of viral DNA synthesis foroptimal expression (Holland et al., 1980).

In line with the complexity of the genome, the life cycle of HSV isquite involved. In addition to the lytic cycle, which results insynthesis of virus particles and, eventually, cell death, the virus hasthe capability to enter a latent state in which the genome is maintainedin neural ganglia until some as of yet undefined signal triggers arecurrence of the lytic cycle. Avirulent variants of HSV have beendeveloped and are readily available for use in gene therapy contexts(U.S. Pat. No. 5,672,344).

E) Vaccinia Virus

Vaccinia virus vectors have been used extensively because of the ease oftheir construction, relatively high levels of expression obtained, widehost range and large capacity for carrying DNA. Vaccinia contains alinear, double-stranded DNA genome of about 186 kb that exhibits amarked “A-T” preference. Inverted terminal repeats of about 10.5 kbflank the genome. The majority of essential genes appear to map withinthe central region, which is most highly conserved among poxviruses.Estimated open reading frames in vaccinia virus number from 150 to 200.Although both strands are coding, extensive overlap of reading frames isnot common.

At least 25 kb can be inserted into the vaccinia virus genome (Smith andMoss, 1983). Prototypical vaccinia vectors contain transgenes insertedinto the viral thymidine kinase gene via homologous recombination.Vectors are selected on the basis of a tk-phenotype. Inclusion of theuntranslated leader sequence of encephalomyocarditis virus, the level ofexpression is higher than that of conventional vectors, with thetransgenes accumulating at 10% or more of the infected cell's protein in24 h (Elroy-Stein et al., 1989).

F) SV40 Virus

Simian virus 40 (SV40) was discovered in 1960 as a contaminant in poliovaccines prepared from rhesus monkey kidney cell cultures. It was foundto cause tumors when injected into newborn hamsters. The genome is adouble-stranded, circular DNA of about 5000 bases encoding large (708AA) and small T antigens (174 AA), agnoprotein and the structuralproteins VP1, VP2 and VP3. The respective size of these molecules is362, 352 and 234 amino acids.

Little is known of the nature of the receptors for any polyoma virus.The virus is taken up by endocytosis and transported to the nucleuswhere uncoating takes place. Early mRNA's initiate viral replication andis necessary, along with DNA replication, for late gene expression. Nearthe origin of replication, promoters are located for early and latetranscription. Twenty-one base pair repeats, located 40–103 nucleotidesupstream of the initiation transcription site, are the main promotingelement and are binding sites for Sp1, while 72 base pair repeats act asenhancers.

Large T antigen, one of the early proteins, plays a critical role inreplication and late gene expression and is modified in a number ofways, including N-terminal acetylation, phosphorylation, poly-ADPribosylation, glycosylation and acylation. The other T antigen isproduced by splicing of the large T transcript. The corresponding smallT protein is not strictly required for infection, but it plays a role inthe accumulation of viral DNA.

DNA replication is controlled, to an extent, by a genetically definedcore region that includes the viral origin of replication. The SV40element is about 66 bp in length and has subsequences of AT motifs, GCmotifs and an inverted repeat of 14 bp on the early gene side. Large Tantigen is required for initiation of DNA replication, and this proteinhas been shown to bind in the vicinity of the origin. It also hasATPase, adenylating and helicase activities.

After viral replication begins, late region expression initiates. Thetranscripts are overlapping and, in some respect, reflect differentreading frames (VP1 and VP2/3). Late expression initiates is the samegeneral region as early expression, but in the opposite direction. Thevirion proteins are synthesized in the cytoplasm and transported to thenucleus where they enter as a complex. Virion assembly also takes placein the nucleus, followed by lysis and release of the infectious virusparticles.

It is contemplated that the present invention will encompass SV40vectors lacking all coding sequences. The region from about 5165–5243and about 0–325 contains all of the control elements necessary forreplication and packaging of the vector and expression of any includedgenes. Thus, minimal SV40 vectors are derived from this region andcontain at least a complete origin of replication.

Because large T antigen is believed to be involved in the expression oflate genes, and no large T antigen is expressed in the target cell, itwill be desired that the promoter driving the heterologous gene be apolyomavirus early promoter, or more preferably, a heterologouspromoter. Thus, where heterologous control elements are utilized, theSV40 promoter and enhancer elements are dispensable.

D) Other Viral Vectors

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such aspapillomaviruses, papovaviruses and lentivirus may be employed. Theseviruses offer several features for use in gene transfer into variousmammalian cells, and it will be understood that various modifications tosuch viruses can be made to enhance for example infectivity andtargeting. Chimeric viruses, employing advantageous portions ofdifferent viruses, may also be constructed by one of skill in the art.

6. Engineering of Viral Vectors

In certain embodiments, the present invention further involves themanipulation of viral vectors. Such methods involve the use of a vectorconstruct containing, for example, a heterologous DNA encoding a gene ofinterest and a means for its expression, replicating the vector in anappropriate helper cell, obtaining viral particles produced therefrom,and infecting cells with the recombinant virus particles. The gene couldsimply encode a protein for which large quantities of the protein aredesired, i.e., large scale in vitro production methods. Alternatively,the gene could be a therapeutic gene, for example to treat cancer cells,to express immunomodulatory genes to fight viral infections, or toreplace a gene's function as a result of a genetic defect. In thecontext of the gene therapy vector, the gene will be a heterologous DNA,meant to include DNA derived from a source other than the viral genomewhich provides the backbone of the vector. Finally, the virus may act asa live viral vaccine and express an antigen of interest for theproduction of antibodies thereagainst. The gene may be derived from aprokaryotic or eukaryotic source such as a bacterium, a virus, a yeast,a parasite, a plant, or even an animal. The heterologous DNA also may bederived from more than one source, i.e., a multigene construct or afusion protein. The heterologous DNA may also include a regulatorysequence which may be derived from one source and the gene from adifferent source.

A) Therapeutic Genes

p53 currently is recognized as a tumor suppressor gene (Montenarh,1992). High levels of mutant p53 have been found in many cellstransformed by chemical carcinogenesis, ultraviolet radiation, andseveral viruses, including SV40. The p53 gene is a frequent target ofmutational inactivation in a wide variety of human tumors and is alreadydocumented to be the most frequently-mutated gene in common humancancers (Mercer, 1992). It is mutated in over 50% of human NSCLC(Hollestein et al., 1991) and in a wide spectrum of other tumors.

The p53 gene encodes a 393-amino-acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are generally minute by comparison with transformed cells or tumortissue. Interestingly, wild-type p53 appears to be important inregulating cell growth and division. Overexpression of wild-type p53 hasbeen shown in some cases to be anti-proliferative in human tumor celllines. Thus, p53 can act as a negative regulator of cell growth(Weinberg, 1991) and may directly suppress uncontrolled cell growth ordirectly or indirectly activate genes that suppress this growth. Thus,absence or inactivation of wild-type p53 may contribute totransformation. However, some studies indicate that the presence ofmutant p53 may be necessary for full expression of the transformingpotential of the gene.

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and are knownto occur in at least 30 distinct codons, often creating dominant allelesthat produce shifts in cell phenotype without a reduction tohomozygosity. Additionally, many of these dominant negative allelesappear to be tolerated in the organism and passed on in the germ line.Various mutant alleles appear to range from minimally dysfunctional tostrongly penetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encodingwild-type p53 into two human breast cancer cell lines restores growthsuppression control in such cells (Casey et al., 1991). A similar effecthas also been demonstrated on transfection of wild-type, but not mutant,p53 into human lung cancer cell lines (Takahasi et al., 1992). p53appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 is not detrimental to normal cellswith endogenous wild-type p53. Thus, such constructs might be taken upby normal cells without adverse effects. It is thus proposed that thetreatment of p53-associated cancers with wild-type p53 expressionconstructs will reduce the number of malignant cells or their growthrate. Furthermore, recent studies suggest that some p53 wild-type tumorsare also sensitive to the effects of exogenous p53 expression.

The major transitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁ phase. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, e.g. p16^(INK4), which has been biochemically characterized asa protein that specifically binds to and inhibits CDK4, and thus mayregulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano, 1993),deletion of this gene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16^(B), p21^(WAF1, CIP1, SDI1), and p27^(KIP1). Thep16^(INK4) gene maps to 9p21, a chromosome region frequently deleted inmany tumor types. Homozygous deletions and mutations of the p16^(INK4)gene are frequent in human tumor cell lines. This evidence suggests thatthe p16^(INK4) gene is a tumor suppressor gene. This interpretation hasbeen challenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994a; Kamb et al., 1994b; Mori etal., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16^(INK4) functionby transfection with a plasmid expression vector reduced colonyformation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

C-CAM is expressed in virtually all epithelial cells (Odin and Obrink,1987). C-CAM, with an apparent molecular weight of 105 kD, wasoriginally isolated from the plasma membrane of the rat hepatocyte byits reaction with specific antibodies that neutralize cell aggregation(Obrink, 1991). Recent studies indicate that, structurally, C-CAMbelongs to the immunoglobulin (Ig) superfamily and its sequence ishighly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti,1989). Using a baculovirus expression system, Cheung et al. (1993a;1993b and 1993c) demonstrated that the first Ig domain of C-CAM iscritical for cell adhesion activity.

Cell adhesion molecules, or CAMs are known to be involved in a complexnetwork of molecular interactions that regulate organ development andcell differentiation (Edelman, 1985). Recent data indicate that aberrantexpression of CAMs may be involved in the tumorigenesis of severalneoplasms; for example, decreased expression of E-cadherin, which ispredominantly expressed in epithelial cells, is associated with theprogression of several kinds of neoplasms (Edelman and Crossin, 1991;Frixen et al., 1991; Bussemakers et al., 1992; Matsura et al., 1992;Umbas et al., 1992). Also, Giancotti and Ruoslahti (1990) demonstratedthat increasing expression of α₅β₁ integrin by gene transfer can reducetumorigenicity of Chinese hamster ovary cells in vivo. C-CAM now hasbeen shown to suppress tumor growth in vitro and in vivo.

Other tumor suppressors that may be employed according to the presentinvention include RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1,p73, BRCA1, VHL, FCC, MMAC1, MCC, p16, p21, p57, pTEN, C-CAM, p27, mda-7and BRCA2. Inducers of apoptosis, such as Bax, Bak, Bcl-X_(s), Bik, Bid,Harakiri, Ad E1B, Bad and ICE-CED3 proteases, similarly could find useaccording to the present invention.

Various enzyme genes are of interest according to the present invention.Such enzymes include cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,α-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinaseand human thymidine kinase.

Hormones are another group of gene that may be used in the vectorsdescribed herein. Included are growth hormone, prolactin, placentallactogen, luteinizing hormone, follicle-stimulating hormone, chorionicgonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin(ACTH), angiotensin I and II, β-endorphin, β-melanocyte stimulatinghormone (β-MSH), cholecystokinin, endothelin I, galanin, gastricinhibitory peptide (GIP), glucagon, insulin, lipotropins, neurophysins,somatostatin, calcitonin, calcitonin gene related peptide (CGRP),β-calcitonin gene related peptide, hypercalcemia of malignancy factor(1–40), parathyroid hormone-related protein (107–139) (PTH-rP),parathyroid hormone-related protein (107–111) (PTH-rP), glucagon-likepeptide (GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM,secretin, vasoactive intestinal peptide (VIP), oxytocin, vasopressin(AVP), vasotocin, enkephalinamide, metorphinamide, alpha melanocytestimulating hormone (alpha-MSH), atrial natriuretic factor (5–28) (ANF),amylin, amyloid P component (SAP-1), corticotropin releasing hormone(CRH), growth hormone releasing factor (GHRH), luteinizinghormone-releasing hormone (LHRH), neuropeptide Y, substance K(neurokinin A), substance P and thyrotropin releasing hormone (TRH).

Other classes of genes that are contemplated to be inserted into thevectors of the present invention include interleukins and cytokines.Interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11 IL-12, GM-CSF and G-CSF.

Examples of diseases for which the present viral vector would be usefulinclude, but are not limited to, adenosine deaminase deficiency, humanblood clotting factor IX deficiency in hemophilia B, and cysticfibrosis, which would involve the replacement of the cystic fibrosistransmembrane receptor gene. The vectors embodied in the presentinvention could also be used for treatment of hyperproliferativedisorders such as rheumatoid arhritis or restenosis by transfer of genesencoding angiogenesis inhibitors or cell cycle inhibitors. Transfer ofprodrug activators such as the HSV-TK gene can be also be used in thetreatment of hyperploiferative disorders, including cancer.

B) Antisense Constructs

Oncogenes such as ras, myc, neu, raf erb, src, fins, jun, trk, ret, gsp,hst, bcl and abl also are suitable targets. However, for therapeuticbenefit, these oncogenes would be expressed as an antisense nucleicacid, so as to inhibit the expression of the oncogene. The term“antisense nucleic acid” is intended to refer to the oligonucleotidescomplementary to the base sequences of oncogene-encoding DNA and RNA.Antisense oligonucleotides, when introduced into a target cell,specifically bind to their target nucleic acid and interfere withtranscription, RNA processing, transport and/or translation. Targetingdouble-stranded (ds) DNA with oligonucleotide leads to triple-helixformation; targeting RNA will lead to double-helix formation.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNAs, maybe employed to inhibit gene transcription or translation or both withina host cell, either in vitro or in vivo, such as within a host animal,including a human subject. Nucleic acid sequences comprising“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,that the larger purines will base pair with the smaller pyrimidines toform only combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA.

As used herein, the terms “complementary” or “antisense sequences” meannucleic acid sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, nucleicacid sequences of fifteen bases in length may be termed complementarywhen they have a complementary nucleotide at thirteen or fourteenpositions with only single or double mismatches. Naturally, nucleic acidsequences which are “completely complementary” will be nucleic acidsequences which are entirely complementary throughout their entirelength and have no base mismatches.

While all or part of the gene sequence may be employed in the context ofantisense construction, statistically, any sequence 17 bases long shouldoccur only once in the human genome and, therefore, suffice to specify aunique target sequence. Although shorter oligomers are easier to makeand increase in vivo accessibility, numerous other factors are involvedin determining the specificity of hybridization. Both binding affinityand sequence specificity of an oligonucleotide to its complementarytarget increases with increasing length. It is contemplated thatoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore base pairs will be used. One can readily determine whether a givenantisense nucleic acid is effective at targeting of the correspondinghost cell gene simply by testing the constructs in vitro to determinewhether the endogenous gene's function is affected or whether theexpression of related genes having complementary sequences is affected.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines. Oligonucleotides which contain C-5 propyneanalogues of uridine and cytidine have been shown to bind RNA with highaffinity and to be potent antisense inhibitors of gene expression(Wagner et al., 1993).

As an alternative to targeted antisense delivery, targeted ribozymes maybe used. The term “ribozyme” refers to an RNA-based enzyme capable oftargeting and cleaving particular base sequences in oncogene DNA andRNA. Ribozymes can either be targeted directly to cells, in the form ofRNA oligo-nucleotides incorporating ribozyme sequences, or introducedinto the cell as an expression construct encoding the desired ribozymalRNA. Ribozymes may be used and applied in much the same way as describedfor antisense nucleic acids.

C) Antigens for Vaccines

Other therapeutics genes might include genes encoding antigens such asviral antigens, bacterial antigens, fungal antigens or parasiticantigens. Viruses include picomavirus, coronavirus, togavirus,flavirviru, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus,arenvirus, reovirus, retrovirus, papovavirus, parvovirus, herpesvirus,poxvirus, hepadnavirus, and spongiform virus. Preferred viral targetsinclude influenza, herpes simplex virus 1 and 2, measles, small pox,polio or HIV. Pathogens include trypanosomes, tapeworms, roundworms,helminths. Also, tumor markers, such as fetal antigen or prostatespecific antigen, may be targeted in this manner. Preferred examplesinclude HIV env proteins and hepatitis B surface antigen. Administrationof a vector according to the present invention for vaccination purposeswould require that the vector-associated antigens be sufficientlynon-immunogenic to enable long term expression of the transgene, forwhich a strong immune response would be desired. Preferably, vaccinationof an individual would only be required infrequently, such as yearly orbiennially, and provide long term immunologic protection against theinfectious agent.

D) Control Regions

In order for the viral vector to effect expression of a transcriptencoding a therapeutic gene, the polynucleotide encoding the therapeuticgene will be under the transcriptional control of a promoter and apolyadenylation signal. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the host cell, or introduced syntheticmachinery, that is required to initiate the specific transcription of agene. A polyadenylation signal refers to a DNA sequence recognized bythe synthetic machinery of the host cell, or introduced syntheticmachinery, that is required to direct the addition of a series ofnucleotides on the end of the mRNA transcript for proper processing andtrafficking of the transcript out of the nucleus into the cytoplasm fortranslation. The phrase “under transcriptional control” means that thepromoter is in the correct location in relation to the polynucleotide tocontrol RNA polymerase initiation and expression of the polynucleotide.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7–20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30–110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, β-actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Examplesof transgenes that may be toxic to the producer cell line arepro-apoptotic and cytokine genes. Several inducible promoter systems areavailable for production of viral vectors where the transgene productmay be toxic.

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.This system is designed to allow regulated expression of a gene ofinterest in mammalian cells. It consists of a tightly regulatedexpression mechanism that allows virtually no basal level expression ofthe transgene, but over 200-fold inducibility. The system is based onthe heterodimeric ecdysone receptor of Drosophila, and when ecdysone oran analog such as muristerone A binds to the receptor, the receptoractivates a promoter to turn on expression of the downstream transgenehigh levels of mRNA transcripts are attained. In this system, bothmonomers of the heterodimeric receptor are constituitively expressedfrom one vector, whereas the ecdysone-responsive promoter which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest wouldtherefore be useful. Cotransfection of plasmids containing the gene ofinterest and the receptor monomers in the producer cell line would thenallow for the production of the gene transfer vector without expressionof a potentially toxic transgene. At the appropriate time, expression ofthe transgene could be activated with ecdysone or muristeron A.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, Calif.) originally developed by Gossen andBujard (Gossen and Bujard, 1992; Gossen et al., 1995). This system alsoallows high levels of gene expression to be regulated in response totetracycline or tetracycline derivatives such as doxycycline. In theTet-On™ system, gene expression is turned on in the presence ofdoxycycline, whereas in the Tet-Off™ system, gene expression is turnedon in the absence of doxycycline. These systems are based on tworegulatory elements derived from the tetracycline resistance operon ofE. coli. The tetracycline operator sequence to which the tetracyclinerepressor binds, and the tetracycline repressor protein. The gene ofinterest is cloned into a plasmid behind a promoter that hastetracycline-responsive elements present in it. A second plasmidcontains a regulatory element called the tetracycline-controlledtransactivator, which is composed, in the Tet-Off™ system, of the VP16domain from the herpes simplex virus and the wild-type tertracyclinerepressor. Thus in the absence of doxycycline, transcription isconstituitively on. In the Tet-On™ system, the tetracycline repressor isnot wild type and in the presence of doxycycline activatestranscription. For gene therapy vector production, the Tet-Off™ systemwould be preferable so that the producer cells could be grown in thepresence of tetracycline or doxycycline and prevent expression of apotentially toxic transgene, but when the vector is introduced to thepatient, the gene expression would be constituitively on.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoetic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

Similarly tissue specific promoters may be used to effect transcriptionin specific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA, probasin, prostatic acid phosphatase or prostate-specificglandular kallikrein (hK2) may be used to target gene expression in theprostate. Similarly, the following promoters may be used to target geneexpression in other tissues (Table 3).

TABLE 3 Tissue specific promoters Tissue Promoter Pancreas Insulinelastin amylase pdr-1 pdx-1 glucokinase Liver albumin PEPCK HBV enhanceralpha fetoprotein apolipoprotein C alpha-1 antitrypsin vitellogenin,NF-AB Transthyretin Skeletal muscle myosin H chain muscle creatinekinase dystrophin calpain p94 skeletal alpha-actin fast troponin 1 Skinkeratin K6 keratin K1 Lung CFTR human cytokeratin 18 (K18) pulmonarysurfactant proteins A, B and C CC-10 P1 Smooth muscle sm22 alphaSM-alpha-actin Endothelium endothelin-1 E-selectin von Willebrand factorTIE (Korhonen et al., 1995) KDR/flk-1 Melanocytes Tyrosinase Adiposetissue lipoprotein lipase (Zechner et al., 1988) adipsin (Spiegelman etal., 1989) acetyl-CoA carboxylase (Pape and Kim, 1989) glycerophosphatedehydrogenase (Dani et al., 1989) adipocyte P2 (Hunt et al., 1986) Bloodβ-globin

In certain indications, it may be desirable to activate transcription atspecific times after administration of the gene therapy vector. This maybe done with such promoters as those that are hormone or cytokineregulatable. For example in gene therapy applications where theindication is a gonadal tissue where specific steroids are produced orrouted to, use of androgen or estrogen regulated promoters may beadvantageous. Such promoters that are hormone regulatable include MMTV,MT-1, ecdysone and RuBisco. Other hormone regulated promoters such asthose responsive to thyroid, pituitary and adrenal hormones are expectedto be useful in the present invention. Cytokine and inflammatory proteinresponsive promoters that could be used include K and T Kininogen(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone etal., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBPalpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson etal., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988),alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988),angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible byphorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin and alpha-1 antichymotrypsin.

It is envisioned that cell cycle regulatable promoters may be useful inthe present invention. For example, in a bi-cistronic gene therapyvector, use of a strong CMV promoter to drive expression of a first genesuch as p16 that arrests cells in the G1 phase could be followed byexpression of a second gene such as p53 under the control of a promoterthat is active in the G1 phase of the cell cycle, thus providing a“second hit” that would push the cell into apoptosis. Other promoterssuch as those of various cyclins, PCNA, galectin-3, E2F1, p53 and BRCA1could be used.

Tumor specific promoters such as osteocalcin, hypoxia-responsive element(HRE), MAGE4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may alsobe used to regulate gene expression in tumor cells. Other promoters thatcould be used according to the present invention includeLac-regulatable, chemotherapy inducible (e.g. MDR), and heat(hyperthermia) inducible promoters, Radiation-inducible (e.g., EGR (Jokiet al., 1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acidpromoters, U1 snRNA (Bartlett et al., 1996), MC-1, PGK, -actin andalpha-globin. Many other promoters that may be useful are listed inWalther and Stein (1996).

It is envisioned that any of the above promoters alone or in combinationwith another may be useful according to the present invention dependingon the action desired. In addition, this list of promoters is should notbe construed to be exhaustive or limiting, those of skill in the artwill know of other promoters that may be used in conjunction with thepromoters and methods disclosed herein. A further list of promoters isprovided in the Table 4.

TABLE 4 PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light ChainT-Cell Receptor HLA DQ α and DQ β β-Interferon Interleukin-2Interleukin-2 Receptor MHC Class II 5 MHC Class II HLA-DRα β-ActinMuscle Creatine Kinase Prealbumin (Transthyretin) Elastase IMetallothionein Collagenase Albumin Gene α-Fetoprotein τ-Globin β-Globinc-fos c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM)α1-Antitrypsin H2B (TH2B) Histone Mouse or Type I CollagenGlucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone HumanSerum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived Growth FactorDuchenne Muscular Dystrophy SV40 Polyoma Retroviruses Papilloma VirusHepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus GibbonApe Leukemia Virus

The promoter further may be characterized as an inducible promoter. Aninducible promoter is a promoter which is inactive or exhibits lowactivity except in the presence of an inducer substance. Some examplesof promoters that may be included as a part of the present inventioninclude, but are not limited to, MT II, MMTV, Colleganse, Stromelysin,SV40, Murine MX gene, α-2-Macroglobulin, MHC class I gene h-2kb, HSP70,Proliferin, Tumor Necrosis Factor, or Thyroid Stimulating Hormone αgene. The associated inducers are shown in Table 5. It is understoodthat any inducible promoter may be used in the practice of the presentinvention and that all such promoters would fall within the spirit andscope of the claimed invention.

TABLE 5 Element Inducer MT II Phorbol Ester (TPA) Heavy metals MMTV(mouse mammary tumor Glucocorticoids virus) B-Interferon poly(rI)Xpoly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ CollagenasePhorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 PhorbolEster (TPA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kBInterferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPATumor Necrosis Factor FMA Thyroid Stimulating Hormone α Thyroid HormoneGene

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter and the Rous sarcoma virus longterminal repeat can be used to obtain high-level expression of thepolynucleotide of interest. The use of other viral or mammalian cellularor bacterial phage promoters which are well-known in the art to achieveexpression of polynucleotides is contemplated as well, provided that thelevels of expression are sufficient to produce a growth inhibitoryeffect.

By employing a promoter with well-known properties, the level andpattern of expression of a polynucleotide following transfection can beoptimized. For example, selection of a promoter which is active inspecific cells, such as tyrosinase (melanoma), alpha-fetoprotein andalbumin (liver tumors), CC10 (lung tumor) and prostate-specific antigen(prostate tumor) will permit tissue-specific expression of thetherapeutic gene.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base (EPDB)) could also be used to drive expression of aparticular construct. Use of a T3, T7 or SP6 cytoplasmic expressionsystem is another possible embodiment. Eukaryotic cells can supportcytoplasmic transcription from certain bacteriophage promoters if theappropriate bacteriophage polymerase is provided, either as part of thedelivery complex or as an additional genetic expression vector.

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Such polyadenylation signals as that fromSV40, bovine growth hormone, and the herpes simplex virus thymidinekinase gene have been found to function well in a number of targetcells.

7. Methods of Gene Transfer

In order to create the helper cell lines of the present invention, andto create recombinant adenovirus vectors for use therewith, variousgenetic (i.e. DNA) constructs must be delivered to a cell. One way toachieve this is via viral transductions using infectious viralparticles, for example, by transformation with an adenovirus vector ofthe present invention. Alternatively, retroviral or bovine papillomavirus may be employed, both of which permit permanent transformation ofa host cell with a gene(s) of interest. In other situations, the nucleicacid to be transferred is not infectious, i.e., contained in aninfectious virus particle. This genetic material must rely on non-viralmethods for transfer.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979), cell sonication(Fechheimer et al., 1987), gene bombardment using high velocitymicroprojectiles (Yang et al., 1990), and receptor-mediated transfection(Wu and Wu, 1987; Wu and Wu, 1988).

Once the construct has been delivered into the cell the nucleic acidencoding the therapeutic gene may be positioned and expressed atdifferent sites. In certain embodiments, the nucleic acid encoding thetherapeutic gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In one embodiment of the invention, the expression construct may simplyconsist of naked recombinant DNA or plasmids. Transfer of the constructmay be performed by any of the methods mentioned above which physicallyor chemically permeabilize the cell membrane. This is particularityapplicable for transfer in vitro, however, it may be applied for in vivouse as well. Dubensky et al. (1984) successfully injected polyomavirusDNA in the form of CaPO₄ precipitates into liver and spleen of adult andnewborn mice demonstrating active viral replication and acute infection.Benvenisty and Neshif (1986) also demonstrated that directintraperitoneal injection of CaPO₄ precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga CAM may also be transferred in a similar manner in vivo and expressCAM.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Using the β-lactamase gene, Wong et al.(1980) demonstrated the feasibility of liposome-mediated delivery andexpression of foreign DNA in cultured chick embryo, HeLa, and hepatomacells. Nicolau et al. (1987) accomplished successful liposome-mediatedgene transfer in rats after intravenous injection. Also included arevarious commercial approaches involving “lipofection” technology.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention.

Other expression constructs which can be employed to deliver a nucleicacid encoding a therapeutic gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a therapeutic genealso may be specifically delivered into a cell type such as prostate,epithelial or tumor cells, by any number of receptor-ligand systems withor without liposomes. For example, the human prostate-specific antigen(Watt et al., 1986) may be used as the receptor for mediated delivery ofa nucleic acid in prostate tissue.

8. Removing Nucleic Acid Contaminants

The present invention employs nucleases to remove contaminating nucleicacids. Exemplary nucleases include Benzonase®, Pulmozyme®; or any otherDNase or RNase commonly used within the art.

Enzymes such as Benzonaze® degrade nucleic acid and have no proteolyticactivity. The ability of Benzonase® to rapidly hydrolyze nucleic acidsmakes the enzyme ideal for reducing cell lysate viscosity. It is wellknown that nucleic acids may adhere to cell derived particles such asviruses. The adhesion may interfere with separation due toagglomeration, change in size of the particle or change in particlecharge, resulting in little if any product being recovered with a givenpurification scheme. Benzonase® is well suited for reducing the nucleicacid load during purification, thus eliminating the interference andimproving yield.

As with all endonucleases, Benzonase® hydrolyzes internal phosphodiesterbonds between specific nucleotides. Upon complete digestion, all freenucleic acids present in solution are reduced to oligonucleotides 2 to 4bases in length.

9. Purification Techniques

The present invention employs a number of different purification topurify adenoviral vectors of the present invention. Such techniquesinclude those based on sedimentation and chromatography and aredescribed in more detail herein below.

A) Density Gradient Centrifugation

There are two methods of density gradient centrifugation, the rate zonaltechnique and the isopycnic (equal density) technique, and both can beused when the quantitative separation of all the components of a mixtureof particles is required. They are also used for the determination ofbuoyant densities and for the estimation of sedimentation coefficients.

Particle separation by the rate zonal technique is based upondifferences in size or sedimentation rates. The technique involvescarefully layering a sample solution on top of a performed liquiddensity gradient, the highest density of which exceeds that of thedensest particles to be separated. The sample is then centrifuged untilthe desired degree of separation is effected, i.e., for sufficient timefor the particles to travel through the gradient to form discrete zonesor bands which are spaced according to the relative velocities of theparticles. Since the technique is time dependent, centrifugation must beterminated before any of the separated zones pellet at the bottom of thetube. The method has been used for the separation of enzymes, hormones,RNA-DNA hybrids, ribosomal subunits, subcellular organelles, for theanalysis of size distribution of samples of polysomes and forlipoprotein fractionations.

The sample is layered on top of a continuous density gradient whichspans the whole range of the particle densities which are to beseparated. The maximum density of the gradient, therefore, must alwaysexceed the density of the most dense particle. During centrifugation,sedimentation of the particles occurs until the buoyant density of theparticle and the density of the gradient are equal (i.e., wherep_(p)=p_(m) in equation 2.12). At this point no further sedimentationoccurs, irrespective of how long centrifugation continues, because theparticles are floating on a cushion of material that has a densitygreater than their own.

Isopyenic centrifugation, in contrast to the rate zonal technique, is anequilibrium method, the particles banding to form zones each at theirown characteristic buoyant density. In cases where, perhaps, not all thecomponents in a mixture of particles are required, a gradient range canbe selected in which unwanted components of the mixture will sediment tothe bottom of the centrifuge tube whilst the particles of interestsediment to their respective isopycnic positions. Such a techniqueinvolves a combination of both the rate zonal and isopycnic approaches.

Isopycnic centrifugation depends solely upon the buoyant density of theparticle and not its shape or size and is independent of time. Hencesoluble proteins, which have a very similar density (e.g., p=1.3 g cm⁻³in sucrose solution), cannot usually be separated by this method,whereas subcellular organelles (e.g., Golgi apparatus, p=1.11 g cm⁻³,mitochondria, p=1.19 g cm⁻³ and peroxisomes, p=1.23 g cm⁻³ in sucrosesolution) can be effectively separated.

As an alternative to layering the particle mixture to be separated ontoa preformed gradient, the sample is initially mixed with the gradientmedium to give a solution of uniform density, the gradient‘self-forming’, by sedimentation equilibrium, during centrifugation. Inthis method (referred to as the equilibrium isodensity method), use isgenerally made of the salts of heavy metals (e.g., caesium or rubidium),sucrose, colloidal silica or Metrizamide.

The sample (e.g., DNA) is mixed homogeneously with, for example, aconcentrated solution of caesium chloride. Centrifugation of theconcentrated caesium chloride solution results in the sedimentation ofthe CsCl molecules to form a concentration gradient and hence a densitygradient. The sample molecules (DNA), which were initially uniformlydistributed throughout the tube now either rise or sediment until theyreach a region where the solution density is equal to their own buoyantdensity, i.e. their isopycnic position, where they will band to formzones. This technique suffers from the disadvantage that often very longcentrifugation times (e.g., 36 to 48 hours) are required to establishequilibrium. However, it is commonly used in analytical centrifugationto determine the buoyant density of a particle, the base composition ofdouble stranded DNA and to separate linear from circular forms of DNA.

Many of the separations can be improved by increasing the densitydifferences between the different forms of DNA by the incorporation ofheavy isotopes (e.g., ¹⁵N) during biosynthesis, a technique used byLeselson and Stahl to elucidate the mechanism of DNA replication inEsherichia coli, or by the binding of heavy metal ions or dyes such asethidium bromide. Isopycnic gradients have also been used to separateand purify viruses and analyze human plasma lipoproteins.

B) Chromatography

In certain embodiments of the invention, it will be desirable to producepurified adenovirus. Purification techniques are well known to those ofskill in the art. These techniques tend to involve the fractionation ofthe cellular milieu to separate the adenovirus particles from othercomponents of the mixture. Having separated adenoviral particles fromthe other components, the adenovirus may be purified usingchromatographic and electrophoretic techniques to achieve completepurification. Analytical methods particularly suited to the preparationof a pure adenovrial particle of the present invention are ion-exchangechromatography, size exclusion chromatography; polyacrylamide gelelectrophoresis. A particularly efficient purification method to beemployed in conjunction with the present invention is HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of anadenoviral particle. The term “purified” as used herein, is intended torefer to a composition, isolatable from other components, wherein theadenoviral particle is purified to any degree relative to itsnaturally-obtainable form. A purified adenoviral particle therefore alsorefers to an adenoviral component, free from the environment in which itmay naturally occur.

Generally, “purified” will refer to an adenoviral particle that has beensubjected to fractionation to remove various other components, and whichcomposition substantially retains its expressed biological activity.Where the term “substantially purified” is used, this designation willrefer to a composition in which the particle, protein or peptide formsthe major component of the composition, such as constituting about 50%or more of the constituents in the composition.

Various methods for quantifying the degree of purification of a proteinor peptide will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificactivity of an active fraction, or assessing the amount of polypeptideswithin a fraction by SDS/PAGE analysis. A preferred method for assessingthe purity of a fraction is to calculate the specific activity of thefraction, to compare it to the specific activity of the initial extract,and to thus calculate the degree of purity, herein assessed by a “-foldpurification number”. The actual units used to represent the amount ofactivity will, of course, be dependent upon the particular assaytechnique chosen to follow the purification and whether or not theexpressed protein or peptide exhibits a detectable activity.

There is no general requirement that the adenovirus, always be providedin their most purified state. Indeed, it is contemplated that lesssubstantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater -fold purification than thesame technique utilizing a low pressure chromatography system. Methodsexhibiting a lower degree of relative purification may have advantagesin total recovery of protein product, or in maintaining the activity ofan expressed protein.

Of course, it is understood that the chromatographic techniques andother purification techniques known to those of skill in the art mayalso be employed to purify proteins expressed by the adenoviral vectorsof the present invention. Ion exchange chromatography and highperformance liquid chromatography are exemplary purification techniquesemployed in the purification of adenoviral particles and are describedin further detail herein below.

Ion-Exchange Chromatography. The basic principle of ion-exchangechromatography is that the affinity of a substance for the exchangerdepends on both the electrical properties of the material and therelative affinity of other charged substances in the solvent. Hence,bound material can be eluted by changing the pH, thus altering thecharge of the material, or by adding competing materials, of which saltsare but one example. Because different substances have differentelectrical properties, the conditions for release vary with each boundmolecular species. In general, to get good separation, the methods ofchoice are either continuous ionic strength gradient elution or stepwiseelution. (A gradient of pH alone is not often used because it isdifficult to set up a pH gradient without simultaneously increasingionic strength.) For an anion exchanger, either pH and ionic strengthare gradually increased or ionic strength alone is increased. For acation exchanger, both pH and ionic strength are increased. The actualchoice of the elution procedure is usually a result of trial and errorand of considerations of stability. For example, for unstable materials,it is best to maintain fairly constant pH.

An ion exchanger is a solid that has chemically bound charged groups towhich ions are electrostatically bound; it can exchange these ions forions in aqueous solution. Ion exchangers can be used in columnchromatography to separate molecules according to charge,; actuallyother features of the molecule are usually important so that thechromatographic behavior is sensitive to the charge density, chargedistribution, and the size of the molecule.

The principle of ion-exchange chromatography is that charged moleculesadsorb to ion exchangers reversibly so that molecules can be bound oreluted by changing the ionic environment. Separation on ion exchangersis usually accomplished in two stages: first, the substances to beseparated are bound to the exchanger, using conditions that give stableand tight binding; then the column is eluted with buffers of differentpH, ionic strength, or composition and the components of the buffercompete with the bound material for the binding sites.

An ion exchanger is usually a three-dimensional network or matrix thatcontains covalently linked charged groups. If a group is negativelycharged, it will exchange positive ions and is a cation exchanger. Atypical group used in cation exchangers is the sulfonic group, SO₃ ⁻. Ifan H⁺ is bound to the group, the exchanger is said to be in the acidform; it can, for example, exchange on H⁺ for one Na⁺ or two H⁺ for oneCa²⁺. The sulfonic acid group is called a strongly acidic cationexchanger. Other commonly used groups are phenolic hydroxyl andcarboxyl, both weakly acidic cation exchangers. If the charged group ispositive—for example, a quaternary amino group—it is a strongly basicanion exchanger. The most common weakly basic anion exchangers arearomatic or aliphatic amino groups.

The matrix can be made of various material. Commonly used materials aredextran, cellulose, agarose and copolymers of styrene and vinylbenzenein which the divinylbenzene both cross-links the polystyrene strands andcontains the charged groups. Table 6 gives the composition of many ionexchangers.

The total capacity of an ion exchanger measures its ability to take upexchangeable groups per milligram of dry weight. This number is suppliedby the manufacturer and is important because, if the capacity isexceeded, ions will pass through the column without binding.

TABLE 6 Matrix Exchanger Functional Group Tradename Dextran StrongCationic Sulfopropyl SP-Sephadex Weak Cationic Carboxymethyl CM-Sephadex Strong Anionic Diethyl-(2- QAE-Sephadex hydroxypropyl)-aminoethyl Weak Anionic Diethylaminoethyl DEAE-Sephadex CelluloseCationic Carboxymethyl CM-Cellulose Cationic Phospho P-cel AnionicDiethylaminoethyl DEAE-cellulose Anionic Polyethylenimine PEI-CelluloseAnionic Benzoylated- DEAE (BND)- naphthoylated, cellulosedeiethylaminoethyl Anionic p-Aminobenzyl PAB-cellulose Styrene- StrongCationic Sulfonic acid AG 50 divinyl- Strong Anionic AG 1-Source15Qbenzene Strong Sulfonic acid + AG 501 Cationic + Tetramethyl- StrongCationic ammonium Acrylic Weak Cationic Carboxylic Bio-Rex 70 StrongAnionic Trimethylamino- E. Merk ethyl Strong Anionic Trimethylamino TosoHaas TSK- group Gel-Q-5PW Phenolic Strong Cationic Sulfonic acid Bio-Rex40 Expoxyamine Weak Anionic Tertiary amino AG-3

The available capacity is the capacity under particular experimentalconditions (i.e., pH, ionic strength). For example, the extent to whichan ion exchanger is charged depends on the pH (the effect of pH issmaller with strong ion exchangers). Another factor is ionic strengthbecause small ions near the charged groups compete with the samplemolecule for these groups. This competition is quite effective if thesample is a macromolecule because the higher diffusion coefficient ofthe small ion means a greater number of encounters. Clearly, as bufferconcentration increases, competition becomes keener.

The porosity of the matrix is an important feature because the chargedgroups are both inside and outside the matrix and because the matrixalso acts as a molecular sieve. Large molecules may be unable topenetrate the pores; so the capacity will decease with increasingmolecular dimensions. The porosity of the polystyrene-based resins isdetermined by the amount of cross-linking by the divinylbenzene(porosity decreases with increasing amounts of divinylbenzene). With theDowex and AG series, the percentage of divinylbenzene is indicated by anumber after an X—hence, Dowex 50-X8 is 8% divinylbenzene.

Ion exchangers come in a variety of particle sizes, called mesh size.Finer mesh means an increased surface-to-volume ration and thereforeincreased capacity and decreased time for exchange to occur for a givenvolume of the exchanger. On the other hand, fine mesh means a slow flowrate, which can increase diffusional spreading. The use of very fineparticles, approximately 10 μm in diameter and high pressure to maintainan adequate flow is called high-performance or high-pressure liquidchromatography or simply HPLC.

Such a collection of exchangers having such different properties—charge,capacity, porosity, mesh—makes the selection of the appropriate one foraccomplishing a particular separation difficult. How to decide on thetype of column material and the conditions for binding and elution isdescribed in the following Examples.

There are a number of choices to be made when employing ion exchangechromatography as a technique. The first choice to be made is whetherthe exchanger is to be anionic or cationic. If the materials to be boundto the column have a single charge (i.e., either plus or minus), thechoice is clear. However, many substances (e.g., proteins, viruses),carry both negative and positive charges and the net charge depends onthe pH. In such cases, the primary factor is the stability of thesubstance at various pH values. Most proteins have a pH range ofstability (i.e., in which they do not denature) in which they are eitherpositively or negatively charged. Hence, if a protein is stable at pHvalues above the isoelectric point, an anion exchanger should be used;if stable at values below the isoelectric point, a cation exchanger isrequired.

The choice between strong and weak exchangers is also based on theeffect of pH on charge and stability. For example, if a weakly ionizedsubstance that requires very low or high pH for ionization ischromatographed, a strong ion exchanger is called for because itfunctions over the entire pH range. However, if the substance is labile,weak ion exchangers are preferable because strong exchangers are oftencapable of distorting a molecule so much that the molecule denatures.The pH at which the substance is stable must, of course, be matched tothe narrow range of pH in which a particular weak exchanger is charged.Weak ion exchangers are also excellent for the separation of moleculeswith a high charge from those with a small charge, because the weaklycharged ions usually fail to bind. Weak exchangers also show greaterresolution of substances if charge differences are very small. If amacromolecule has a very strong charge, it may be impossible to elutefrom a strong exchanger and a weak exchanger again may be preferable. Ingeneral, weak exchangers are more useful than strong exchangers.

The Sephadex and Bio-gel exchangers offer a particular advantage formacromolecules that are unstable in low ionic strength. Because thecross-links in these materials maintain the insolubility of the matrixeven if the matrix is highly polar, the density of ionizable groups canbe made several times greater than is possible with cellulose ionexchangers. The increased charge density means increased affinity sothat adsorption can be carried out at higher ionic strengths. On theother hand, these exchangers retain some of their molecular sievingproperties so that sometimes molecular weight differences annul thedistribution caused by the charge differences; the molecular sievingeffect may also enhance the separation.

Small molecules are best separated on matrices with small pore size(high degree of cross-linking) because the available capacity is large,whereas macromolecules need large pore size. However, except for theSephadex type, most ion exchangers do not afford the opportunity formatching the porosity with the molecular weight.

The cellulose ion exchangers have proved to be the best for purifyinglarge molecules such as proteins and polynucleotides. This is becausethe matrix is fibrous, and hence all functional groups are on thesurface and available to even the largest molecules. In may caseshowever, beaded forms such as DEAE-Sephacel and DEAE-Biogel P are moreuseful because there is a better flow rate and the molecular sievingeffect aids in separation.

Selecting a mesh size is always difficult. Small mesh size improvesresolution but decreases flow rate, which increases zone spreading anddecreases resolution. Hence, the appropriate mesh size is usuallydetermined empirically.

Because buffers themselves consist of ions, they can also exchange, andthe pH equilibrium can be affected. To avoid these problems, the rule ofbuffers is adopted: use cationic buffers with anion exchangers andanionic buffers with cation exchangers. Because ionic strength is afactor in binding, a buffer should be chosen that has a high bufferingcapacity so that its ionic strength need not be too high. Furthermore,for best resolution, it has been generally found that the ionicconditions used to apply the sample to the column (the so-calledstarting conditions) should be near those used for eluting the column.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

10. Quality Control Assays

Recombinant adenovirus vectors made according to the present inventionare tested to ensure that they meet desired product releasespecifications. These specifications are defined by assays forbiological activity, virus titer, final product purity, identity andphysico-chemical characteristics. These assays are performed at variousstages of production including analysis of the crude cell lysate,in-process bulk (pre-filter), in-process bulk (post-filter), and thefmal product. Crude cell lysate is defined as the material that isremoved from the cell culture appartus before any processing has beendone. In-process bulk (prefilter) is defined as the material that hasbeen processed through the HPLC purification step, but has not beensterile filtered prior to vialing. In-process bulk (post filter) isdefined as the material that has been sterile filtered and is ready tobe vialed. Final product is defined as the material that has been placedinto individual vials and is ready for storage or use. It will beunderstood that similar protocols may be used as tests for Ad5CMV-p53 aswell as other adenoviral vectors containing the same or differenttransgenes. The following section describes representative assays usedfor testing the recombinant adenovirus product.

A. Safety Assays

General Safety Assay

The general safety assay test (C.F.R. 610.11) is performed to detect thepresence of extraneous toxic contaminants. Guinea pigs (Hartley albino,either sex) and mice (Swiss outbred, either sex) are inoculatedintraperitoneally with the test article diluted in sterile water forinjection and observed for overt signs of ill health, weight loss, ordeath for the test period. Their weights are measured just prior to andupon completion of the test period of 7 days. A passing test is one inwhich the controls perform as expected and the animals inoculated withthe test article have satisfactory responses.

PCR Assay for the Detection of Adeno-Associated Virus (AAV) inBiological Samples

This assay detects the presence of AAV nucleic acid sequences by PCRamplification with a set of primers targeted to a conserved region inthe capsid gene. The amplified DNA from the test article is run on anagarose gel containing ethidium bromide and visualized by photography.Briefly, the DNA is extracted from the test sample, and 0.5 microgramsis analyzed by PCR. PCR amplification is performed using AAVoligonucleitides primers specific for the capsid region of AAV. Negativeand positive control DNA is also analyzed. Assay acceptance isdetermined by the absence of any bands in the negative control sample,and the expected size band in the positive control sample. For thepresent assay, a specific 459 bp band is the expected size. A passingtest for the test article is the absence of the 459 base pair band.

In Vitro Assay for the Presence of Viral Contaminants

This assay determines whether adventitious viral contaminants arepresent in the test article through the inoculation and observation ofthree types of indicator cells. The presence of viral contamination isdetermined by observations for cytopathic effect (CPE) or other visuallydiscernible effects, hemagglutination, and hemadsorption. The indicatorcells include MRC-5, a diploid human lung line; Vero, an African greenmonkey kidney line; and HeLa, a human epithelioid carcinoma cell line.Briefly, the three indicator cell lines are seeded into 6-well platesand maintained for approximately 24 hours. The cultures are theninoculated with 0.5 ml of the adenoviral sample or virus controls andallowed to absorb for 1 hours at 36 degrees Celsius. The virus is thenremoved and replaced with culture medium, and the wells observed for 14days for evidence of CPE. Each well is also tested for hemadsorption andhemagglutination using three types of erythrocytes. All culture fluidsare blind passaged onto additional culture plates of indicator cells andobserved for CPE for another 14 days.

To accept this assay, certain criteria should preferably be met. Theseinclude: 1) each of the positive control viruses should preferably causeCPE in the indicator cell lines into which it is inoculated; 2) each ofthe positive control viruses should preferably produce hemadsorptionand/or hemagglutination with at least one type of erythrocyte at 4degrees Celsius and/or 36 degrees Celsius at one or more time pointswith each of the indicator cells lines into which it is inoculated; 3)The indicator cells lines inoculated with the negative control shouldpreferably not exhibit any CPE, hemadsorption, or hemagglutination. Apassing test for the test article is preferably the absence of CPE,hemadsorption and hemagglutination.

In Vivo Adventitious Virus Assay

This assay is designed to detect the presence of viruses which do notcause a discernible effect in in vitro cell culture systems, but maycause unwanted effects in vivo. The experimental design utilizesinoculations of adult and suckling mice, guinea pigs, and embryonatedhens' eggs, and is similar to that used by the British Institute forBiological Standards and Control. This test includes blind passages ofhomogenates to successive animals and/or hens eggs to increase thelikelihood of detection of low level viral contaminants.

The test method is as follows. Suckling mice will be inoculatedintraperitoneally, per os, and intracranially and observed for 14 days.A single pool of emulsified tissue (minus skin and gastrointestinaltract) of all surviving mice will be used to inoculate additional miceusing the same routes. Sham control mice will also be inoculated. Adultmice of both sexes will be inoculated intraperitoneally, per os,intradermally, and intracranially and observed for 28 days. Shamcontrols will also be inoculated. Adult guinea pigs of both sexes willbe inoculated intraperitoneally and intracranially and observed for 28days. Sham control guinea pigs will also be inoculated. The yolk sac of6–7 days old embryonated hens′ eggs will be inoculated and incubated atleast nine days. The yolk sacs will be harvested, pooled, and a 10%suspension will be sub-passaged into new embryonated hens' eggs. Ninedays later, the eggs are evaluated for viability.

Acceptance criteria for the assay include healthy animals at the startof the testing, and the tests will be considered valid if about 90% ofcontrol adult mice, about 80% of control suckling mice, about 80% of theembryonated hens' eggs, and about 75% of the control guinea pigs survivethe incubation period and show no lesions at the site of inoculation orshow no signs of viral infection. The test article will be considerednot contaminated if about 80% of the animals remain healthy and survivethe observation period and if about 95% the animals used in the testfail to show any lesions of any kind at the site of injection and failto show any signs of viral infection.

B. Purity Assays

BCA Assay for Total Protein

This assay allows for a quantitative determination of total protein inthe final product. The assay uses the Pierce BCA kit procedure. Briefly,replicate samples are prepared and placed in a microtiter plate. ABovine Serum Albumin (BSA) standard is prepared and placed in amicrotiter plate as a control. For a negative control, diluent is placedin a microtiter plate. The BCA reagent is dispensed into the microtiterplates and the plates are incubated to allow color development. Theplates are then read spectrophotometrically at 550 nm, and the testsample concentrations are calculated based on the BSA standard.Preferred protein content by BCA is 250 to 500 micrograms per 1×10e12viral particles. Most preferable protein content is 260 to 320micrograms per 1×10e12 viral particles. The protein concentrationdetermined by this assay is used to calculate the amount of protein toload on the SDS-PAGE gel for restriction analysis.

Sterility Assay

Sterility assays (documented in U.S.P. XXIII <71>) are used at both thebulk and final product stage. Sterility testing is via membranefiltration and is performed in a soft-wall isolator system to minimizelaboratory contamination of samples tested. All test articles shouldpreferably pass the sterility test.

Bioburden Test

The bioburden test is used to detect microbial load in a test sample byfiltering the test sample onto a membrane filter, placing the membranefilter onto Tryptic Soy agar and Sabourad agar plates and observing forgrowth after 2–5 days incubation. Suspensions with known levels ofBacillus subtilis and Candid albicans are also assayed to confirm assaysuitability.

Briefly the test method is as follows. Test samples may be stored up to24 hours at 2–8 degree Celsius before testing. Reserve samples that arenot to be tested within 24 hours may be frozen at less than −60 degreesCelsius. Negative controls (sterile diluent) are prepared by filtering100 mL of sterile diluent through an analytical filter unit using avacuum. The membrane filter is removed from the unit and placed on apre-warmed Tryptic Soy agar plate. The process is repeated using asecond filter unit and the filter is placed on a pre-warmed Sabouraudagar plate. In-process test samples are tested by filtering 5×10 mL ofcrude cell lysate onto 5 separate filters or 10 mL of prefiltered bulkproduct onto a single filter. Each membrane filter is removed from theunit and placed on a pre-warmed Tryptic Soy agar plate. The process isrepeated using a second set of filter units and the filter is placed ona pre-warmed Sabouraud agar plate. Bacillus subtilis positive controlsare prepared by filtering 50 mL of sterile diluent through an analyticalfilter unit using a vacuum. The membrane filter is removed from the unitand placed on a pre-warmed Tryptic Soy agar plate. The process isrepeated using a Candida albicans positive control using a second filterunit and the filter is placed on a pre-warmed Sabouraud agar plate.Tryptic Soy agar plates are incubated at 30–35 degrees Celsius for 2–5days. Sabouraud agar plates are incubated at 22–27 degrees Celsius for5–7 days. Colonies on the membrane filters are counted after theincubation period. The assays are acceptable when the negative controlsexhibit no growth and positive controls exhibit 1–100 colonies permembrane filter. The test article should preferably contain less than orequal to 1000 colony forming units per 100 mL of the crude cell lysate.It is more preferable that the crude cell lysate contain less than orequal to 500 colony forming units per 100 mL, and most preferable thatthe crude cell lysate contain less than or equal to 10 colony formingunits per 100 mL. It is most preferable that the prefiltered bulkproduct contain less than or equal to one colony forming unit per 10 mL.Using purification techniques in accordance with the present disclosure,bioburden values less than 1 have been obtained at the crude cell lysatestep, and less than 1 at the prefiltered bulk product step.

Bacterial Endotoxin Test

The purpose of this test is to measure the amount of gram negativebacterial endotoxin in a given sample. The Limulus Amebocyte Lysate(LAL) assay is performed in accordance with USPXXIII using a commercialchromogenic test kit. It is used to quantify the gram-negative bacterialendotoxin level in test samples. Dilutions of samples are run with andwithout a spike of endotoxin for evaluation of inhibition or enhancementeffects.

The test method is performed according to the directions outlined in thetest kit insert, and is as follows. The assay is performed in 96 wellplates and LAL-free water is used as an assay blank. A standard curveranging from 0.01 to 5.0 endotoxin units/mL is made using commerciallyavailable exdotoxin standard. Test samples are tested either neat ordiluted appropriately in endotoxin free water. Positive controls areprepared by spiking test samples at each dilution with 0.05 EU/mL. Allmanipulations are performed in pyrogen free glass or polystyrene tubesusing pyrogen free pipette tips. The 96 well plate is incubatred withblank, standard curve, test samples, and positive control for 10minutes, after which the LAL reagent is added to each well. The plate isread in a kinetic reader at 405 nm for 150 seconds and the results areexpressed in EU/ML.

For the assay to be acceptable, the standard curve should preferably belinear with an r value of −0.980 to 1.000, the slope of the curve shouldpreferably be −0.0. to −0.100, the Y-intercept should preferably be2.5000 to 3.5000 and endotoxin recovery in the positive control shouldpreferably be 5–150% of the spike. It is preferable that the sample haveless than five (5) EU/mL, more preferably the the sample have less than3 EU/mL, and most preferable that the sample have less than 0.05 EU/mL.Using purification techniques in accordance with the present disclosure,endotoxin values as low as 0.15 have been obtained at the prefilteredbulk product step and as low as 0.3 at the final product step.

Test for the Presence of Agar-Cultivable and Non-Cultivable Mycoplasmas

This assay detects the presence of Mycoplasma in a test article based onthe ability of Mycoplasma to grow in any one of the test systems: Agarisolation and Vero cell culture system. Growth is signified by colonyformation, shift in pH indicators, or presence of Mycoplasma bystaining, depending on the system used. The assay is performed using alarge sample volume. The test methods are as follows. The test articleand positive controls are inoculated directly onto Mycoplasma agarplates and into Mycoplasma semi-solid broth which is subcultured threetimes onto agar plates. The samples are incubated both aerobically andanaerobically. At 14 days post-infection the agar plates are examinedfor evidence of growth. The test article is also inoculated directlyonto Vero cell cultures and incubated for 3–5 days. The cultures arestained with a DNA-binding fluorochrome and evaluated microscopically byepifluorescence for the presence of Mycoplasma.

For the Agar isolation assay, the positive controls should preferablyshow Mycoplasma growth in at least two out of five direct plates foreach media type and for each incubation condition, and in thesemi-broth. The negative control plates and bottles should preferablyshow absence of Mycoplasma growth. For the Vero cell culture assay,positive controls should preferably show the presence of Mycoplasma,negative controls should preferably show no presence of Mycoplasma, andall of the controls should preferably show the absence of bacterial orfungal contaminants. The test article will preferably be negative forthe presence of Mycoplasma.

Contaminating Host Cell DNA Assay

This method allows evaluation of contaminating host cell DNA in a finalproduct. Test samples are extracted and examined for contaminating DNA.The test method is as follows. Samples are extracted and transferred tonitrocellulose. Diluted reference samples are spiked with human DNA andtransferred to nitrocellulose. Positive controls are prepared by spikinghuman DNA into aliquots of BSA and transferred to nitrocellulose. Thenitrocellulose with all samples and controls is probed with a³²P-labeled human DNA probe. The filter is rinsed and the hybridizedradioactivity is measured using an AMBIS Radioanalytic Imaging System.Acceptable performance of the assay is determined by the controlsperforming as expected, and a test article should preferably have lessthan or equal to 10 ng contaminating host cell DNA per 1×10¹² viralparticles. It is more preferable that the level of contaminating humanDNA be less than 7 ng/1×10¹² viral particles, even more preferable thatthe level of contaminating human DNA be less than 5 ng/1×10¹² viralparticles, even more preferable that the level of contaminating humanDNA be less than 3 ng/1×10¹² viral particles and most preferable thatthe level of contaminating human DNA be less than 5 pg/1×10¹² viralparticles. Using purification techniques in accordance with the presentdisclosure, contaminating DNA values as low as 200 pg/mL have beenobtained at the final product step and 80 pg/mL in a developmentalbatch.

Quantitative Replication Competent Adenovirus (RCA) Assay.

The RCA present in a recombinant-defective adenovirus population such asAd5CMV-p53 are detected by infection of non-competent A549 humancarcinoma cells. A549 cells are grown in cell culture dishes to give amonolayer of cells and are then infected with the adenovirus sample tobe tested. After 4 hours of infection time, the supernatant is discardedand the A549 monolayer is covered by a mixture containing both culturemedia and agarose. After solidification, the agarose limits any infectedcell to formation of a single plaque. After 14 days at 37 degreesCelsius, agarose is stained with neutral red and the visualized plaquesare counted. Positive controls are run concomitantly and contain eitherwild type adenovirus alone or the test article spiked with wild typeadenovirus such that any inhibitory effect coming from the sample couldbe detected. In order to characterize any observed RCA, all plaques aresubcultured and PCR characterized. PCR analysis is performed usingprobes targeted against the E1 region in order to demonstrate thepresence of E1 region in the vector, and against the E3 region toexclude the presence of wild type viruses. It has been demonstrated thatthe presence of E1 excludes the presence of the p53 gene and that theRCA consist of only double homologous constructions.

The test methodology is as follows. A human lung carcinoma line, A549,is grown to sub-confluence in cell culture dishes and then infected withthe Ad5CMV-p53 sample to be tested at an MOI of less than 200 viralparticles per cell. The cells are then exposed to the virus for a 4 hourinfection time, the supernatant is discarded, and the cell monolayer iscovered with a medialagarose overlay. One positive control containingwild type adenovirus and one containing the test sample spiked with wildtype adenovirus are run concomitantly to assure assay sensitivity. Aftera 14 day incubation at 37 degrees Celsius the overlay is stained withneutral red to allow visualization of any plaques. Plaques are counted,picked and transferred to 0.8 mL of culture media and subjected to threefreeze-thaw cycles to release virus. The plaque supernatant is then usedto infect additional multi well dishes of A549 cells. The cells areobserved for CPE and the supernatant from those dishes is harvested. Theharvested supernatant is subjected to amplification by PCR using probesdirected against the E1 region of the wild type adenovirus genome andagainst the E3 region of the wild type adenovirus virus. If the E3region is present the RCA is scored as wild type. If only the E1 regionis present the RCA is scored as a double homologous recombinationproduct. For the assay to be considered valid, all controls must performas expected. It is preferable that the test article contain less than 40plaque forming units in 1×10¹¹ viral particles. It is more preferablethat the test article contain less than 4 plaque forming units in 1×10¹¹viral particles, and most preferable that the test article contain lessthan 0.4 plaque forming units in 1×10¹¹ viral particles. Usingpurification techniques in accordance with the present disclosure, RCAvalues ≦1 in 2.5×10¹¹ virus particles have been obtained at the finalproduct step.

Determination of BSA Levels

This assay is used to determine levels of contaminating bovine serumalbumin (BSA) in adenoviral preparations. In certain recombinantadenovirus production runs, the vector is produced in a cell culturesystem containing bovine serum. This assay is an enzyme linkedimmunosorbent assay (ELISA) that detects the presence and quantity oflow levels of BSA that remain in the final product.

The test method is as follows. A standard curve ranging from 1.9 ng to1125 ng/mL of purified BSA is prepared. A positive control is preparedby spiking 0.2% gelatin with 3.9, 15, and 62.5 ng/mL BSA. A negativecontrol sample is 0.2% gelatin in Tris buffered saline. The test sampleis tested neat and at dilutions of 1:10 through 1:320. All samples andcontrols are transferred to an ELISA assay plate, and the BSA content isdetected with a probe antibody specific for BSA. The plates are read at492 nm. For the assay to be considered valid, the blank OD₄₉₂ shouldpreferably be less than 0.350. The test article should preferablycontain less than 100 ng BSA per 1×10¹² viral particles. It is morepreferable that the test article contain less than 85 ng BSA per 1×10¹²viral particles, even more preferable that the test article contain lessthan 75 ng BSA per 1×10¹² vi particles, even more preferable that thetest article contain less than 65 ng BSA per 1×10¹² viral particles andmost preferable that the test article contain less than 1 ng BSA per1×10¹² viral particles. Using purification techniques in accordance withthe present disclosure, BSA values <1.9 ng/1×10¹² virus particles havebeen obtained at the final product step.

P53 Mutation Assay

This assay is to demonstrate the ability of p53 expressed fromAd5CMV-p53 final product to activate transcription. The criticalbiochemical function of p53, which underlies its tumor suppressoractivity, is the ability to activate transcription. Mutant proteins failto activate transcription in mammalian cells. The transcriptionalactivity of human p53 is conserved in yeast, and mutant which areinactive in human cells are also inactive in yeast. The detection of p53mutations is possible in yeast by testing the transcriptional competenceof human p53 expressed in a Saccharomyces cerevisiae defective inadenine synthesis due to a mutation in ADE2 but which contains a secondcopy of ADE2 in an open reading frame controlled by a p53 responsivepromoter. The Saccharomyces cerevisiae strain is cotransformed with alinearized plasmid and the isolated p53 fragment from Ad5CMV-p53.Recombinants will constituitively express p53. When grown on adeninepoor media, the yeast strain will appear red. If the yeast carries awild-type p53 gene the colonies will appear white.

The test method is as follows. DNA from the test article is extractedusing a phenol/chloroform/isoamyl alcohol procedure and the p53 DNAinsert from the adenoviral genome is isolated following restrictiondigestion. An expression vector containing the ADH1 promoter islinearized. Yeast (strain yIG397) is co-transformed with the DNAfragment bearing the p53 gene from the test article and the linearizedexpression vector. A p53 expression vector is formed in vivo byhomologous recombination. The yeast cultures are grown for two to threedays at 30 degrees Celsius. The ADH1 promoter causes recombinants toconstituitively express p53. The yIG397 strain of yeast is defective inadenine synthesis because of a mutation in the endogenous ADE2 gene, butit contains a second copy of the ADE2 open reading frame controlled bythe p53-responsive ADH1 promoter. The colonies of yIG397 that are ADE2mutant turn red when grown on low adenine plates. Colonies of yIG397with mutant p53 are also red, and colonies containing wild type p53 arewhite. Red and white colonies are counted at the end of the assay. Theassay is considered valid if all the controls perform as expected, andthe test article should preferably contain p53 mutations at a frequencyof less than 3% to pass product release specifications. It is morepreferable that the test article contain less than 2% p53 mutations, andmost preferable that the test article contain 0% p53 mutations. Usingpurification techniques in accordance with the present disclosure, p53mutations values ≦1% have been obtained at the final product step.

Plaque Assay for Adenoviral Vectors

This assay is used to determine the titer of adenoviral material in thefinal product by measuring the development of plaques on human 293cells, which are derived from human embryonic kidney. Ad5CMV-p53 isreplication deficient on normal cells due to deletion of the E1 region.The E1 function is provided in trans in 293 cells which contain the E1region of adenovirus type 5. Five fold dilutions of the test article areutilized to quantify the titer.

The test method is as follows. Human 293 cells are seeded in 66 welltissue culture plates and the cells are allowed to grow to greater than90% confluence before infection. Vector dilutions are made to target5–80 plaques per well. A reference virus is used as a control. Twoconcentrations are tested for the positive control using six replicates.Four concentrations are tested for each sample using six replicates. Thevector is allowed to infect for one hour during which the plates arerocked every 15 minutes to ensure even coverage of the virus. After theincubation period, the cells are overlaid with a 0.5% agarose solution,and the virus-infected cells are incubated for six days at which timethey are stained with Neutral Red. The plaques are counted between fourand 25 hours after staining, depending on the size of the plaques. Wellswhich contain greater than 80 plaques are scored TNTC (Too Numerous ToCount), and wells that cannot be counted are marked as NC (Not Counted)and the reason is noted on the record. Plaque counts and theirrespective dilutions are used to calculate the sample titer. For theassay to be considered valid, the negative control wells shouldpreferably contain no plaques, the titer of the positive control shouldpreferably be within one quarter (0.25) log of the official titer of thevirus being used as the positive control, the % CV for the positivecontrol should preferably be less than or equal to 25%, and for any onedilution in the positive control, there should preferably be no morethan three wells designated “NC”.

At the final product testing step, the test article should preferablyhave a titer of 1×10⁷ to 1×10¹² pfu/mL. It is more preferable to have atiter of 1×10⁹ to 1×10¹² pfu/mL, even more preferable to have a titer of1×10¹⁰ to 1×10¹² pfu/mL, even more preferable to have a titer of 5×10¹⁰to 1×10¹² pfu/mL, and most preferable to have a titer of 8×10¹⁰ to1×10¹² pfu/ml. Using purification techniques in accordance with thepresent disclosure, titer values as high as 5×10¹² virus particles/mLhave been obtained at the final product step.

Determination of Viral Particle Concentration and Particle/PFU Ratio

This assay measures the concentration, in viral particles/mL, for asample of adenoviral material. This assay is a spectrophotometric assaythat determines the total number of particles in a sample based onabsorbance at 260 nm. The extinction coefficient used to convert toviral particles is 1 OD₂₆₀=10¹² viral particles.

The test method is as follows. Three replicates are prepared for eachsample using an appropriate dilution to fall within the linear range ofthe spectrophotometer. The virus sample is combined with 1% SDS (or 10%SDS for dilute test samples) and water to achieve a total volume of 150microliters. The sample is incubated at room temperature for 15–30minutes to disrupt the virion. Each sample is read at A₂₆₀ and A₂₈₀ andthe mean optical density for replicate samples is multiplied by thedilution factor to determine viral particles/mL. The Particle/PFU ratiois determined using the titer determined by the plaque assay describedpreviously. For the assay to be considered valid, the % CV for the threesample replicates should preferably be less than or equal to 10%. Thetest sample should preferably contain 1×10⁷ to 2×10¹³ viral particles/mLat the final product step. It is more preferable that the test samplecontain between about 0.8×10¹² and 2×10¹³ viral particles/mL, and mostpreferable that the sample contain between about 1.2×10¹² and 2×10¹³particles/mL It is most preferable that the A₂₆₀/A₂₈₀ is 1.2 to 1.4. Itis preferable that the Particle/PFU ratio is less than 100, even morepreferable that it is less than 75, and most preferable that it is 10 to60. Using purification techniques in accordance with the presentdisclosure, viral particle concentration values as high as 5×10¹² virusparticles/mL have been obtained at the final product step.

Adenoviral p53 Bioactivity Assay

The SAOS LM assay is a bioactivity assay which is conducted for thepurpose of determining the activity of the p53 component of Ad5CMV-p53.The assay measures the inhibition of growth of SAOS-LM cells (humanosteocarcinoma cell line with a homozygous p53 deletion). Anysignificant loss of inhibitory activity compared with a standard wouldindicate the presence of an unacceptable amount of inactive vector. Theinhibition of growth of SAOS cells is followed using the Alamar Blueindicator dye, which is used to quantitatively measure cellproliferation. This dye contains a colorimetric oxidation/reduction(REDOX) indicator. As cellular activity results in chemical reduction ofthe cellular environment, inhibition of growth results in an oxidizedenvironment that allows the measurement of p53 activity.

The test method is as follows. SAOS cells are plated in 96 well platesand grown overnight at 37 degrees Celsius to greater than 75%confluence. Media is removed from the wells and the cells are challengedwith either a media control, positive control virus (MOI=1000) orvarying dilutions of the test sample. Following challenge, the cells areincubated at 37 degrees Celsius for four days. Alamar Blue is added tothe wells and the plates are incubated approximately eight hours at 37degrees Celsius. Cell density is determined by reading the plates at 570nm. To accept the assay the OD₅₇₀ of the positive control must be lessthan 0.1 and the media control cell density must be at least 75%confluent. It is preferable that the MOI of the test article that causes50% cell death is less than 1000 viral particles. It is more preferablethat the test article have an MOI that causes 50% cell death of lessthan 700 viral particles, and most preferable that the MOI that causes50% cell death is less than 400 viral particles. Using purificationtechniques in accordance with the present disclosure, bioactivity valuesas high as 250–260 have been obtained at the final product step.

HPLC Assay for p53.

This assay is a quantitative evaluation of Ad5CMV-p53 particle numberand purity of in-process samples and of final product stability samples.The method allows quantitation of Ad5CMV-p53 particles by an ionexchange HPLC method.

The test method is as follows. A Toso Haas TSK-Gel-Q-5PW column is usedwith a buffered salt gradient mobile phase for separation of virusparticles and impurities. A reference control calibration curve is runon a newly installed column and scanned at A₂₆₀. A blank is prepared andrun using the same column and method. The sample to be tested isprepared by dilution with the same low salt buffer used in gradientformation. The sample absorbance is detected at 260 and 280 nmwavelengths, and the total are for all peaks detected is determined. Theratio of the area for the A₂₆₀/A₂₈₀ peak is determined, and theconcentration for the 260 nm peak is determined by comparison to thereference calibration curve. Assay acceptance criteria include similarprofile to historical samples, and a A₂₆₀/A₂₈₀ ratio of 1.3+/−0.1 Thetest sample should preferably have a purity of greater than or equal to98%. It is more preferable that the purity be greater than 99%, and mostpreferable that the purity is greater than 99.9%. Using purificationtechniques in accordance with the present disclosure, virus purityvalues as high as 99.8% have been obtained at the final product step.

C. Identity Assays

Restriction Enzyme Mapping Assay for Ad5CMV-p53

This method allows evaluation of Ad5CMV-p53 DNA by restriction enzymeanalysis. Restriction enzymes recognize specific base pair sequences onDNA, cutting the DNA at these restriction sites. There are a limitednumber of recognition sites within a vector for any particularrestriction enzyme. Test sample DNA is digested with two restrictionenzymes and the fragments separated electrophoretically in an agarosegel matrix. The DNA fragments are checked for number and size.

The test method is as follows. DNA is extracted from vector particlesusing a commercially available ion exchange spin column. The extractedDNA is quantified and checked for purity by analyzing the A₂₆₀/A₂₈₀ratio. Approximately 0.4–5 micrograms of the extracted DNA is digestedwith a cocktail of two restriction enzymes, Eco RI and Cla I. Thedigested DNA is loaded onto a 1% agarose gel containing ethidium bromidealongside an equal amount of unrestricted DNA from the same sample. Thesamples are separated by electrophoresis and visualized using anultraviolet light source. Data is captured by photography. The assayacceptance criteria that should preferably be met for the assay to beconsidered valid is a A₂₆₀/A₂₈₀ ratio of extracted DNA of greater than1.6. The test article should preferably have restriction fragment sizesthat match the theoretical fragment sizes expected from the sequence ofAd5CMV-p53. The expected band sizes are 486, 2320, 8494 and 24008 basepairs.

SDS Page Assay

This method allows evaluation of total proteins in final product rangingin size from 5 to 100 kDa by separation according to molecular weight.

The test method is as follows. Total proteins are determined using aPierce BCA method according to the protocol described previously in thissection. The test sample, internal standard and molecular weightstandards are prepared in sample buffer and denatured by heating. Allsamples and standards are loaded into wells of a pre-cast Tris-glycinegel and set in an electrophoresis tank containing running buffer. Thegel is run on a constant current setting for approximately 90 minutes.The gel is then removed from the cassette, stained using CoomassieBrilliant Blue stain and destained. The gel is then analyzed using adensitometric scanning instrument, and the data captured by photography.Alternatively, the gel is dried for archiving. In all controls, thepresence of expected proteins is preferable and there should preferablybe no contaminating proteins. In the test sample, the expected bandsshould preferably be observed, with no significant extra bands.

Western Blot Assay:

This method tests for the presence of p53 protein in Ad5CMV-p53transduced cells. The test method is as follows. Individual 60 mm tissueculture dishes for product samples and control samples are seeded at adensity of 7×10⁵ cells and grown to greater than 80% confluence. Thetest article is diluted in media to provide 3.5×10⁸ viral particles/mL.A reference control is diluted to 3.5×10⁸ vp/mL and a negative controlwith no vector is also prepared. The cells are exposed to mediacontaining product for one hour during which the plates are rocked toensure even distribution of vector. At the end of the hour, additionalmedia is added to the dishes and they are incubated for approximatelyfive hours to allow time for expression of p53. At the end of theincubation period, the cells are treated with trypsin to allow harvest,washed with DPBS and solubilized with a detergent buffer. The totalamount of protein in each sample and control is determined by acolorimetric quantitation method (Pierce BCA). For each sample andmethod, 3–5 micrograms of protein are loaded onto a gel alongside acommercially purchased p53 protein reference and separated bypolyacrylamide gel electrophoresis (PAGE). The proteins in the gel aretransferred to a PVDF membrane and the membrane is exposed to a milkbuffer to block non-specific binding sites and then sequentially exposedto antibodies. The primary antibody, a mouse anti-human p53 antibodyspecifically binds to p53. The secondary antibody is a goat anti-mouseIgG with horseradish peroxidase (HRP) covalently bound. A colorimetricsubstrate is exposed to the bound HRP enzyme which enables visualizationof p53 protein on the blot. For the assay to be considered valid, thecontrol p53 band should preferably be visible, and the negative controlshould preferably show no expression of p53. The test article shouldpreferably show expression of p53.

Recoverable Fill Volume Assay

This method is a gravimetric determination of volume recoverable fromthe container closure for Ad5CMV-p53 final product. Product is recoveredfrom seven vials using tared 3 cc syringes and 21G 1.5 inch needles. Theproduct is weighed, and the weights are converted to volume using thespecific gravity of the product of 1.03 g/mL. The balance calibrationmust be met before weighing of the samples and it is preferable that allseven vials tested must meet specification of 1.0 to 1.4 mL ofrecoverable fill volume. It is more preferable that the recoverable fillvolume be 1.1 to 1.3 mL. It will be understood by those of skill in theart that this assay is an example for the Ad5CMVp53 product, and thatthose of skill in the art will be able to modify this assay for otherproducts in other types of container closures.

Physical Description Assay

This method allows evaluation of the physical description of finalproduct. Approximately seven milliliters of product are pooled in aclear plastic tube. The product is inspected by an analyst to documentthe color, transparency, and the presence of any gross particulatematter. The test article should preferably be clear to opalescent andcontain no gross particulate matter by visual inspection.

pH Assay

This method is a pH determination of the Ad5CMV-p53 final product.Approximately 0.5 mL of the product is placed in a tube. The pH isdetermined using a calibrated pH meter at a temperature of 25+/−5degrees Celsius. The pH standard solutions should preferably demonstratea slope range of 80–120%. The pH of the final product should preferablybe between about 6 and about 9. It is more preferable that the pH isbetween about 6.5 and about 8.8, even more preferable that the pH isbetween about 7.0 and about 8.6, and most preferable that the pH isbetween about 7.5 and about 8.5.

Restriction Enzyme Mapping for Identity Testing of Master Viral Bank orWorking Viral Bank

The goal of this test is to assess the identity of the Ad5CMV-p53 genomethrough measurement of the DNA fragments generated after cleavage of thewhole viral genome (approximately 35308 base pairs). When the unpurifiedviruses are contained in a cell mixture such as a virus bank, the viralDNA first has to be extracted from the crude cell lysate. An aliquot ofthe sample is digested by proteinase K in the presence of SDS. The DNAis then extracted using a mixture of phenol/chloroform/isoamyl alcoholand precipitated with ethanol. The DNA concentration is measured by UVspectrometry. Approximately one microgram of the viral DNA is thensubmitted to restriction enzyme digestion. Four individual digests areperformed utilizing a battery of three restriction enzymes in differentcombinations. The digests and DNA size markers are then separated on anagarose gel using electrophoresis and stained with Syb-Green. The gelsare integrated using a camera and a calibration curve calculated fromthe standards. The size of the fragments greater than 500 bp and lessthan 8000 bp is then determined. The size of the fragments obtainedshould preferably correspond to the theoretical size of the fragmentsobtained from the expected theoretical sequence. The fragment sizes ofthe test sample should preferably correspond to those expected from theDNA sequence.

PCR to Detect E1 DNA Sequences in 293 MCB and WCB

This assay is used to determine the identity of the 293 Master andWorking Cell Banks by demonstrating the presence of the E1 region. Usingtwo specific pairs of PCR primers, one targeted against the E1 regionpresent in both 293 cells and wild-type adenovirus and another onetargeted against the E1 region only present in the wild type adenovirus.The method should demonstrate the identity of the 293 cell linecontained in the test article.

The test method is as follows. After thawing, cells from the testarticle are grown using standard conditions in a cell culture dish untila monolayer of cells is obtained. The cells are then digested withproteinase K to remove the proteins, and DNA isolated usingphenol/chloroform/isoamyl alcohol extractions followed by ethanolprecipitation. The extracted DNA is quantified and checked for purity byan absorbance scan from OD260–OD280. The PCR reaction is performed usingthe two E1 targeted pairs of primers on the test article and on bothpositive and negative DNA controls. The negative control is a mammaliancell line which does not contain the E1 region. The positive control isa wild type adenovirus. The PCR products from each reaction are loadedonto an agarose gel and the size of the fragments obtained afterelectrophoresis and staining are recorded using photography. Thenon-bearing E1 mammalian cell line must exhibit no amplification productwith both pairs of PCR primers, while the wild type adenovirus must showthe correct amplification product with both pairs of PCR primers. Thetest article, must demonstrate the correct amplification product withthe pair of primers located in the E1 region described to be present inthe 293 cell, and must be negative with the second pair of primers knownonly to be present in the wild type adenoviral genome.

11. Pharmaceutical Compositions and Formulations

When purified according to the methods set forth above, it iscontemplated that the viral particles of the present invention may beadministered in vitro, ex vivo or in vivo. Thus, it will be desirable toprepare the complex as a pharmaceutical composition appropriate for theintended application. Generally this will entail preparing apharmaceutical composition that is essentially free of pyrogens, as wellas any other impurities that could be harmful to humans or animals. Onealso will generally desire to employ appropriate salts and buffers torender the complex stable and allow for complex uptake by target cells.

Aqueous compositions of the present invention comprise an effectiveamount of the expression construct and nucleic acid, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions can also be referred to as inocula. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The viral particles of the present invention may include classicpharmaceutical preparations for use in therapeutic regimens, includingtheir administration to humans. Administration of therapeuticcompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration will be by orthotopic, intradermal subcutaneous,intramuscular, intraperitoneal, or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions that include physiologically acceptablecarriers, buffers or other excipients. For application against tumors,direct intratumoral injection, inject of a resected tumor bed, regional(i.e., lymphatic) or general administration is contemplated. It also maybe desired to perform continuous perfusion over hours or days via acatheter to a disease site, e.g., a tumor or tumor site.

The therapeutic compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain about 100 mg of human serum albumin permilliliter of phosphate buffered saline. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like may be used.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate.Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial agents, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto well known parameters.

Additional formulations which are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

An effective amount of the therapeutic agent is determined based on theintended goal, for example (i) inhibition of tumor cell proliferation,(ii) elimination or killing of tumor cells, (iii) vaccination, or (iv)gene transfer for long term expression of a therapeutic gene. The term“unit dose” refers to physically discrete units suitable for use in asubject, each unit containing a predetermined-quantity of thetherapeutic composition calculated to produce the desired responses,discussed above, in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theresult desired. Multiple gene therapeutic regimens are expected,especially for adenovirus.

In certain embodiments of the present invention, an adenoviral vectorencoding a tumor suppressor gene will be used to treat cancer patients.Typical amounts of an adenovirus vector used in gene therapy of canceris 10³–10¹⁵ viral particles/dose, (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹³, 10¹⁴, 10¹⁵) wherein the dose may be divided intoseveral injections at different sites within a solid tumor. Thetreatment regimen also may involve several cycles of administration ofthe gene transfer vector over a period of 3–10 weeks. Administration ofthe vector for longer periods of time from months to years may benecessary for continual therapeutic benefit.

In another embodiment of the present invention, an adenoviral vectorencoding a therapeutic gene may be used to vaccinate humans or othermammals. Typically, an amount of virus effective to produce the desiredeffect, in this case vaccination, would be administered to a human ormammal so that long term expression of the transgene is achieved and astrong host immune response develops. It is contemplated that a seriesof injections, for example, a primary injection followed by two boosterinjections, would be sufficient to induce an long term immune response.A typical dose would be from 10⁶ to 10¹⁵ PFU/injection depending on thedesired result. Low doses of antigen generally induce a strongcell-mediated response, whereas high doses of antigen generally inducean antibody-mediated immune response. Precise amounts of the therapeuticcomposition also depend on the judgment of the practitioner and arepeculiar to each individual.

12. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

A) Cells

293 cells (human epithelial embryonic kidney cells) from the Master CellBank were used for the studies.

B) Media

Dulbecco's modified Eagle's medium (DMEM, 4.5 g/L glucose) +10% fetalbovine serum (FBS) was used for the cell growth phase. For the virusproduction phase, the FBS concentration in DMEM was lowered to 2%.

C) Virus

AdCMVp53 is a genetically engineered, replication-incompetent human type5 adenovirus expressing the human wild type p53 protein under control ofthe cytomegalovirus (CMV) immediate early promoter.

D) Celligen Bioreactor

A Celligen bioreactor (New Brunswick Scientific, Co. Inc.) with 5 Ltotal volume (3.5 L working volume) was used to produce virussupernatant using microcarrier culture. 13 g/L glass coated microcarrier(SoloHill) was used for culturing cells in the bioreactor.

E) Production of Virus Supernatant in the Celligen Bioreactor

293 cells from master cell bank (MCB) were thawed and expanded intoCellfactories (Nunc). Cells were generally split at a confluence ofabout 85–90%. Cells were inoculated into the bioreactor at aninoculation concentration of 1×10⁵ cells/ml. Cells were allowed toattach to the microcarriers by intermittent agitation. Continuousagitation at a speed of 30 rpm was started 6–8 hr post cell inoculation.Cells were cultured for 7 days with process parameters set at pH=7.20,dissolved oxygen (DO)=60% of air saturation, temperature=37° C. On day8, cells were infected with AdCMVp53 at an MOI of 5. Fifty hr post virusinfection, agitation speed was increased from 30 rpm to 150 rpm tofacilitate cell lysis and release of the virus into the supernatant. Thevirus supernatant was harvested 74 hr post-infection. The virussupernatant was then filtered for further concentration/diafiltration.

F) Cellcube™ Bioreactor System

A Cellcube™ bioreactor system (Corning-Costar) was also used for theproduction of AdCMVp53 virus. It is composed of a disposable cellculture module, an oxygenator, a medium recirculation pump and a mediumpump for perfusion. The cell culture module used has a culture surfacearea of 21,550 cm² (1 mer).

G) Production of Virus in the Cellcube™

293 cells from master cell bank (MCB) were thawed and expanded intoCellfactories (Nunc). Cells were generally split at a confluence ofabout 85–90%. Cells were inoculated into the Cellcube™ according to themanufacturer's recommendation. Inoculation cell densities were in therange of 1–1.5×10⁴/cm². Cells were allowed to grow for 7 days at 37° C.under culture conditions of pH=7.20, DO=60% air saturation. Mediumperfusion rate was regulated according to the glucose concentration inthe Cellcube™. One day before viral infection, medium for perfusion waschanged from DMEM+10% FBS to DMEM+2% FBS. On day 8, cells were infectedwith AdCMVp53 virus at a multiplicity of infection (MOI) of 5. Mediumperfusion was stopped for 1 hr immediately after infection then resumedfor the remaining period of the virus production phase. Culture washarvested 45–48 hr post-infection.

H) Lysis Solution

Tween-20 (Fisher Chemicals) at a concentration of 1% (v/v) in 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=7.50 buffer was used to lyse cells atthe end of the virus production phase in the Cellcube™.

I) Clarification and Filtration

Virus supernatant from the Celligen bioreactor and virus solution fromthe Cellcube™ were first clarified using a depth filter (Preflow,GelmanSciences), then was filtered through a 0.8/0.22 μm filter(SuporCap 100, GelmanSciences).

J) Concentration/Diafiltration

Tangential flow filtration (TFF) was used to concentrate and bufferexchange the virus supernatant from the Celligen bioreactor and thevirus solution from the Cellcube™. A Pellicon II mini cassette(Millipore) of 300 K nominal molecular weight cut off (NMWC) was usedfor the concentration and diafiltration. Virus solution was firstconcentrated 10-fold. This was followed by 4 sample volume of bufferexchange against 20 mM Tris+1.0 M NaCl+1 mM MgCl₂, pH=9.00 buffer usingthe constant volume diafiltration method.

Similar concentration/diafiltration was carried out for the columnpurified virus. A Pellicon II mini cassette of 100 K NMWC was usedinstead of the 300 K NMWC cassette. Diafiltration was done against 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 buffer or Dulbecco's phosphatebuffered saline (DPBS).

K) Benzonase Treatment

The concentrated/diafiltrated virus solution was treated with Benzonase™(American International Chemicals) at a concentration of 100 u/ml, roomtemperature overnight to reduce the contaminating nucleic acidconcentration in the virus solution.

L) CsCl Gradient Ultracentrifugation

Crude virus solution was purified using double CsCl gradientultracentrifugation using a SW40 rotor in a Beckman ultracentrifuge(XL-90). First, 7 ml of crude virus solution was overlaid on top of astep CsCl gradient made of equal volume of 2.5 ml of 1.25 g/ml and 1.40g/ml CsCl solution, respectively. The CsCl gradient was centrifuged at35,000 rpm for 1 hr at room temperature. The virus band at the gradientinterface was recovered. The recovered virus was then further purifiedthrough a isopicnic CsCl gradient. This was done by mixing the virussolution with at least 1.5-fold volume of 1.33 g/ml CsCl solution. TheCsCl solution was centrifuged at 35,000 rpm for at least 18 hr at roomtemperature. The lower band was recovered as the intact virus. The viruswas immediately dialyzed against 20 mM Tris+1 mM MgCl₂, pH=7.50 bufferto remove CsCl. The dialyzed virus was stored at −70° C. for future use.

M) Ion exchange Chromatography (IEC) Purification

The Benzonase treated virus solution was purified using IEC. Stronganionic resin Toyopearl SuperQ 650M (Tosohaas) was used for thepurification. A FPLC system (Pharmacia) with a XK16 column (Pharmacia)were used for the initial method development. Further scale-up studieswere carried out using a BioPilot system (Pharmacia) with a XK 50 column(Pharmacia). Briefly, the resin was packed into the columns andsanitized with 1 N NaOH, then charged with buffer B which was followedby conditioning with buffer A. Buffers A and B were composed of 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 and 20 mM Tris+2M NaCl+1 MM MgCl₂,pH=9.00, respectively. Viral solution sample was loaded onto theconditioned colum, followed by washing the column with buffer A untilthe UV absorption reached base line. The purified virus was eluted fromthe column by using a 10 column volume of linear NaCl gradient.

N) HPLC Analysis

A HPLC analysis procedure was developed for evaluating the efficiency ofvirus production and purification. Tris(hydroxymethyl)aminomethane(tris) was obtained from FisherBiotech (Cat# BP154-1; Fair Lawn, N.J.,U.S.A.); sodium chloride (NaCl) was obtained from Sigma (Cat# S-7653,St. Louis, Mo., U.S.A.). Both were used directly without furtherpurification. HPLC analyses were performed on an Analytical GradientSystem from Beckman, with Gold Workstation Software (126 binary pump and168 diode array detector) equipped with an anion-exchange column fromTosoHaas (7.5 cm×7.5 mm ID, 10 μm particle size, Cat# 18257). A 1-mlResource Q (Pharmacia) anion-exchange column was used to evaluate themethod developed by Huyghe et al. using their HEPES buffer system. Thismethod was only tried for the Bioreactor system.

The buffers used in the present HPLC system were Buffer A: 10 mM trisbuffer, pH 9.0. Buffer B: 1.5 M NaCl in buffer A, pH 9.0. The bufferswere filtered through a 0.22 μm bottle top filter by Corning (Cat#25970-33). All of the samples were filtered through a 0.8/0.22 μmAcrodisc PF from Gelman Sciences (Cat# 4187) before injection.

The sample is injected onto the HPLC column in a 60–100 μl volume. Afterinjection, the column (TosoHaas) is washed with 20% B for 3 min at aflow rate of 0.75 ml/min. A gradient is then started, in which B isincreased from 20% to 50% over 6 min. Then the gradient is changed from50% to 100% B over 3 min, followed by 100% B for 6 min. The saltconcentration is then changed back stepwise to 20% again over 4 min, andmaintained at 20% B for another 6 min. The retention time of the Adp53is 9.5±0.3 min with A₂₆₀/A₂₈₀≅1.26±0.03. Cleaning of the column aftereach chromatographic run is accomplished by injecting 100 μl of 0.15 MNaOH and then running the gradient.

Example 2 Effect of Medium Perfusion Rate in Cellcube™ on VirusProduction and Purification

For a perfusion cell culture system, such as the Cellcube™, mediumperfusion rate plays an important role on the yield and quality ofproduct. Two different medium perfusion strategies were examined. Onestrategy was to keep the glucose concentration in the Cellcube™≧2 g/L(high perfusion rate). The other one was to keep the glucoseconcentration ≧1 g/L (low medium perfusion rate).

No significant changes in the culture parameters, such as pH, DO, wasobserved between the two different perfusion rates. Approximatelyequivalent amount of crude viruses (before purification) were producedafter harvesting using 1% Tween-20 lysis solution as shown in Table 7.However, dramatic difference was seen on the HPLC profiles of the viralsolutions from the high and low medium perfusion rate production runs.

TABLE 7 Effect of medium glucose concentration on virus yield Glucoseconcentration (g/L) ≧2.0 ≧1.0 Crude virus yield (PFU) 4 × 10¹² 4.9 ×10¹²

As shown in FIG. 1, a very well separated virus peak (retention time9.39 min) was produced from viral solution using low medium perfusionrate. It was found that virus with adequate purity and biologicalactivity was attained after a single step ion exchange chromatographicpurification of the virus solution produced under low medium perfusionrate. On the other hand, no separated virus peak in the retention timeof 9.39 min was observed from viral solution produced using high mediumperfusion rate. This suggests that contaminants which have the sameelution profile as the virus were produced under high medium perfusionrate. Although the nature of the contaminants is not yet clear, it isexpected that the contaminants are related to the increasedextracellular matrix protein production under high medium perfusion rate(high serum feeding) from the producer cells. This poor separationcharacteristic seen on the HPLC created difficulties for process IECpurification as shown in the following Examples. As a result, mediumperfusion rate used during the cell growth and the virus productionphases in the Cellcube™ has a significant effect on the downstream IECpurification of the virus. Low medium perfusion rate is recommended.This not only produces easy to purify crude product but also offers morecost-effective production due to the reduced medium consumption.

Example 3 Methods of Cell Harvest and Lysis

Based on previous experience, the inventors first evaluated thefreeze-thaw method. Cells were harvested from the Cellcube™ 45–48 hrpost-infection. First, the Cellcube™ was isolated from the culturesystem and the spent medium was drained. Then, 50 mM EDTA solution waspumped into the Cube to detach the cells from the culture surface. Thecell suspension thus obtained was centrifuged at 1,500 rpm (BeckmanGS-6KR) for 10 min. The resultant cell pellet was resuspended inDulbecco's phosphate buffered saline (DPBS). The cell suspension wassubjected to 5 cycles of freeze/thaw between 37° C. water bath anddry-ice ethanol bath to release virus from the cells. The crude celllysate (CCL) thus generated was analyzed on HPLC.

FIG. 2 shows the HPLC profile. No virus peak is observed at retentiontime of 9.32 min. Instead, two peaks at retention times of 9.11 and 9.78min are produced. This profile suggests that the other contaminantshaving similar elution time as the virus exist in the CCL and interferewith the purification of the virus. As a result, very low purificationefficiency was observed when the CCL was purified by IEC using FPLC.

In addition to the low purification efficiency, there was a significantproduct loss during the cell harvest step into the EDTA solution asindicated in Table 8. Approximately 20% of the product was lost into theEDTA solution which was discarded. In addition, about 24% of the crudevirus product is present in the spent medium which was also discarded.Thus, only 56% of the crude virus product is in the CCL. Furthermore,freeze-thaw is a process of great variation and very limitedscaleability. A more efficient cell lysis process with less product lossneeded to be developed.

TABLE 8 Loss of virus during EDTA harvest of cells from Cellcube ™ WasteEDTA Crude product Total crude Spent harvest Crude cell product Mediumsolution lysate (PFU) Volume (ml) 2800 2000 82 — Titer (PFU/ml) 2.6 ×10⁸  3 × 10⁸    2 × 10¹⁰ — Total virus 7.2 × 10¹¹ 6 × 10¹¹ 1.64 × 10¹² 3× 10¹² (PFU) Percentage 24% 20% 56% Data was generated from 1 merCellcube ™.

TABLE 9 Evaluation of non-ionic detergents for cell lysis ConcentrationsDetergents (w/v) Chemistry Comments Thesit   1% Dodecylpoly(ethyleneLarge 0.5% glycol ether)_(n), Precipitate 0.1% n = 9–10 NP-40   1%Ethylphenolpoly(ethylene- Large 0.5% glycolether)_(n) precipitate 0.1% n= 9–11 Tween-20   1% Poly(oxyethylene_(n)- Small 0.5%sorbitan-monolaurate precipitate 0.1% n = 20 Brij-58   1%Cetylpoly(ethylene- Cloudy 0.5% glycolether)_(n) Solution 0.1% n = 20Triton X-100   1% Octylphenolpoly(ethylene- Large 0.5% glycolether)_(n)precipitate 0.1% n = 10

Detergents have been used to lyse cells to release intracellularorganelles. Consequently, the inventors evaluated the detergent lysismethod for the release of adenovirus. Table 9 lists the 5 differentnon-ionic detergents that were evaluated for cell lysis. Cells wereharvested from the Cellcube™ 48 hr post-infection using 50 mM EDTA. Thecell pellet was resuspended in the different detergents at variousconcentrations listed in Table 9.

Cell lysis was carried out at either room temperature or on ice for 30min. Clear lysis solution was obtained after centrifugation to removethe precipitate and cellular debris. The lysis solutions were treatedwith Benzonase and then analyzed by HPLC. FIG. 3 shows the HPLC profilesof lysis solutions from the different detergents. Thesit and NP-40performed similarly as Triton X-100. Lysis solution generated from 1%Tween-20 gave the best virus resolution with the least virus resolutionbeing observed with Brij-58. More efficient cell lysis was found atdetergent concentration of 1% (w/v). Lysis temperature did notcontribute significantly to the virus resolution under the detergentconcentrations examined. For the purpose of process simplicity, lysis atroom temperature is recommended. Lysis solution composed of 1% Tween-20in 20 mM Tris+0.25M NaCl+1 mM MgCl₂, pH=7.50 was employed for cell lysisand virus harvest in the Cellcube™.

Example 4 Effects of Concentration/Diafiltration on Virus Recovery

Virus solution from the lysis step was clarified and filtered beforeconcentration/diafiltration. TFF membranes of different NMWCs, including100K, 300K, 500K, and 1000K, were evaluated for efficientconcentration/diafiltration. The highest medium flux with minimal virusloss to the filtrate was obtained with a membrane of 300K NMWC. BiggerNMWC membranes offered higher medium flux, but resulted in greater virusloss to the filtrate, while smaller NMWC membranes achieved aninsufficient medium flux. Virus solution was first concentrated 10-fold,which was followed by 4 sample volumes of diafiltration against 20 mMTris+0.25 M NaCl+1 mM MgCl₂, pH=9.00 buffer using the constant volumemethod. During the concentration/diafiltration process, pressure dropacross the membrane was kept ≦5 psi. Consistent, high level virusrecovery was demonstrated during the concentration/diafiltration step asindicated in Table 10.

TABLE 10 Concentration/diafiltration of crude virus solution Titer(PFU/ml) Volume (ml) Total virus (PFU) Recovery Run #1 Run #2 Run #1 Run#2 Run #1 Run #2 Run #1 Run #2 Before 2.6 × 10⁹ 2 × 10⁹ 1900 2000 4.9 ×10¹²   4 × 10¹² conc./diafl. Post  2.5 × 10¹⁰ 1.7 × 10¹⁰  200 200   5 ×10¹² 3.4 × 10¹² 102% 85% conc./diafl. Conc. 9.5 10 Factor Filtrate   5 ×10⁵ 1 × 10⁶ 3000 3000 1.5 × 10⁹   3 × 10⁹

Example 5 Effect of Salt Addition on Benzonase Treatment

Virus solution after concentration/diafiltration was treated withBenzonase (nuclease) to reduce the concentration of contaminatingnucleic acid in virus solution. Different working concentrations ofBenzonase, which included 50, 100, 200, 300 units/ml, were evaluated forthe reduction of nucleic acid concentrations. For the purpose of processsimplicity, treatment was carried out at room temperature overnight.Significant reduction in contaminating nucleic acid that is hybridizableto human genomic DNA probe was seen after Benzonase treatment.

Table 11 shows the reduction of nucleic acid concentration before andafter Benzonase treatment. Virus solution was analyzed on HPLC beforeand after Benzonase treatment. As shown in FIG. 4A and FIG. 4B, dramaticreduction in the contaminating nucleic acid peak was observed afterBenzonase treatment. This is in agreement with the result of the nucleicacid hybridization assay. Because of the effectiveness, a Benzonaseconcentration of 100 u/ml was employed for the treatment of the crudevirus solution.

TABLE 11 Reduction of contaminating nucleic acid concentration in virussolution Before Treatment After Treatment Reduction Contaminating 200μg/ml 10 ng/ml 2 × 10⁴-fold nucleic acid concentration

-   -   Treatment condition: Benzonase concentration: 100 u/ml,        temperature: room temperature, time: overnight.

Considerable change in the HPLC profile was observed pre- andpost-Benzonase treatment. No separated virus peak was detected atretention time of 9.33 min after Benzonase treatment. At the same time,a major peak with high 260 nm adsorption at retention time of 9.54 minwas developed. Titer assay results indicated that Benzonase treatmentdid not negatively affect the virus titer and virus remained intact andinfectious after Benzonase treatment. It was reasoned that cellularnucleic acid released during the cell lysis step interacted with virusand either formed aggregates with the virus or adsorbed onto the virussurface during Benzonase treatment.

To minimize the possible nucleic acid virus interaction during Benzonasetreatment, different concentrations of NaCl was added into the virussolution before Benzonase treatment. No dramatic change in the HPLCprofile occurred after Benzonase treatment in the presence of 1 M NaClin the virus solution. FIG. 5 shows the HPLC profile of virus solutionafter Benzonase treatment in the presence of 1M NaCl. Unlike that shownin FIG. 4B, virus peak at retention time of 9.35 min still exists postBenzonase treatment. This result indicates that the presence of 1M NaClprevents the interaction of nucleic acid with virus during Benzonasetreatment and facilitates the further purification of virus fromcontaminating nucleic acid.

Example 6 Ion Exchange Chromatographic Purification

The presence of negative charge on the surface of adenovirus atphysiological pH conditions prompted evaluation of anionic ionexchangers for adenovirus purification. The strong anionic ion exchangerToyopearl Super Q 650M was used for the development of a purificationmethod. The effects of NaCl concentration and pH of the loading buffer(buffer A) on virus purification was evaluated using the FPLC system.

A) Method Development

For ion exchange chromatography, buffer pH is one of the most importantparameters and can have dramatic influence on the purificationefficiency. In reference to the medium pH and conductivity used duringvirus production, the inventors formulated 20 mM Tris+1 mM MgCl₂+0.2MNaCl, pH=7.50 as buffer A. A XK16 column packed with Toyopearl SuperQ650M with a height of 5 cm was conditioned with buffer A.

A sample of 5 ml of Benzonase treated concentrated/diafiltrated virussupernatant from the Celligen bioreactor was loaded onto the column.After washing the column, elution was carried out with a linear gradientof over 10 column volumes to reach buffer B (20 mM Tris+1 nM MgCl₂+2MNaCl, pH=7.50).

FIG. 6 shows the elution profile. Three peaks were observed duringelution without satisfactory separation among them. Control studyperformed with 293 cell conditioned medium (with no virus) showed thatthe first two peaks are virus related. To further improve the separationefficiency, the effect of buffer pH was evaluated. Buffer pH wasincreased to 9.00 while keeping other conditions constant. Much improvedseparation, as shown in FIG. 7, was observed as compared to that ofbuffer pH of 7.50. Fractions #3, #4, and #8 were analyzed on HPLC.

As shown in FIG. 8, the majority of virus was found in fraction #4, withno virus being detected in fractions #3 and #8. Fraction #8 was found tobe mainly composed of contaminating nucleic acid. However, thepurification was still not optimal. There is overlap between fractions#3 and #4 with contaminants still detected in fraction #4.

Based on the chromatogram in FIG. 7, it was inferred that furtherimprovement of virus purification could be achieved by increasing thesalt concentration in buffer A. As a result, the contaminants present inthe fraction #3, which is prior to the virus peak, can be shifted to theflow through faction. The NaCl concentration in buffer A was increasedto 0.3 M while keeping other conditions constant. FIG. 9 shows theelution profile under the condition of 0.3 M NaCl in buffer A.

Dramatic improvement in purification efficiency was achieved. Asexpected the contaminant peak observed in FIG. 7 was eliminated underthe increased salt condition. Samples from crude virus sup, flowthrough, peak #1, and peak #2 were analyzed on HPLC and the results areshown in FIG. 10. No virus was detected in the flow through fraction.The majority of the contaminants present in the crude material werefound in the flow through. HPLC analysis of peak #1 showed a single welldefined virus peak. This HPLC profile is equivalent to that obtainedfrom double CsCl gradient purified virus. Peaks observed at retentiontimes of 3.14 and 3.61 min in CsCl gradient purified virus are glycerolrelated peaks. The purified virus has a A260/A280 ratio of 1.27±0.03.This similar to the value of double CsCl gradient purified virus as wellas the results reported by Huyghe et al. (1996). Peak #2 is composedmainly of contaminating nucleic acid. Based on the purification result,the inventors proposed the following method for IEC purification ofadenovirus sup from the bioreactor.

-   -   Buffer A: 20 mM Tris+1 mM MgCl₂+0.3M NaCl, pH=9.00    -   Buffer B: 20 mM Tris+1 mM MgCl₂+2M NaCl, pH=9.00    -   Elution: 10 column volume linear gradient

B) Method Scale-Up

Following the development of the method, purification was scaled-up fromthe XK16 column (1.6 cm I.D.) to a XK50 column (5 cm I.D., 10-foldscale-up) using the same purification method. A similar elution profilewas achieved on the XK50 column as shown in FIG. 11. The virus fractionwas analyzed on HPLC, which indicated equivalent virus purity to thatobtained from the XK16 column.

During the scale-up studies, it was found that it was more convenientand consistent to use conductivity to quantify the salt concentration inbuffer A. The optimal conductivity of buffer A is in the range of 25±2mS/cm at approximately room temperature (21° C.). Samples producedduring the purification process together with double CsCl purified viruswere analyzed on SDS-PAGE.

As shown in FIG. 12, all the major adenovirus structure proteins aredetected on the SDS-PAGE. The IEC purified virus shows equivalentstaining as that of the double CsCl purified virus. Significantreduction in bovine serum albumin (BSA) concentration was achievedduring purification. The BSA concentration in the purified virus wasbelow the detection level of the western blot assay as shown in FIG. 13.

The reduction of contaminating nucleic acid concentration in virussolution during the purification process was determined using nucleicacid slot blot. ³²P labeled human genomic DNA was used as thehybridization probe (because 293 cells are human embryonic kidneycells). Table 12 shows the nucleic acid concentration at differentstages of the purification process. Nucleic acid concentration in thefinal purified virus solution was reduced to 60 pg/ml, an approximate3.6×10⁶-fold reduction compared to the initial virus supernatant. Virustiter and infectious to total particle ratio were determined for thepurified virus and the results were compared to that from double CsClpurification in Table 11. Both virus recovery and particle/PFU ratio arevery similar between the two purification methods. The titer of thecolumn purified virus solution can be further increased by performing aconcentration step.

TABLE 12 Removal of contaminating nucleic acids during purificationContaminating nucleic acid Steps during purification concentration Virussupernatant from bioreactor 220 μg/ml Concentrated/diafiltrated sup 190μg/ml Sup post Benzonase treatment (O/N, RT,  10 ng/ml 100 u/ml)Purified virus from column 210 pg/ml Purified virus post  60 pg/mlconcentration/diafiltration CsCl purified virus 800 pg/ml

Example 7 Other Purification Methods

In addition to the strong anionic ion exchange chromatography, othermodes of chromatographic methods, were also evaluated for thepurification of AdCMVp53 virus (e.g. size exclusion chromatography,hydrophobic interaction chromatography, cation exchange chromatography,or metal ion affinity chromatography). Compared to the Toyopearl SuperQ, all those modes of purification offered much less efficientpurification with low product recovery. Therefore, Toyopearl Super Qresin is recommended for the purification of AdCMVp53. However, otherquaternary ammonium chemistry based strong anionic exchangers are likelyto be suitable for the purification of AdCMVp53 with some processmodifications.

Example 8

Purification of Crude AdCMVp53 Virus Generated from Cellcube™

Two different production methods were developed to produce AdCMVp53virus. One was based on microcarrier culture in a stirred tankbioreactor. The other was based on a Cellcube™ bioreactor. As describedabove, the purification method was developed using crude virussupernatant generated from the stirred tank bioreactor. It was realizedthat although the same medium, cells and viruses were used for virusproduction in both the bioreactor and the Cellcube™, the culture surfaceonto which cells attached was different.

In the bioreactor, cells were grown on a glass coated microcarrier,while in the Cellcube™ cells were grown on proprietary treatedpolystyrene culture surface. Constant medium perfusion was used in theCellcube™, on the other hand, no medium perfusion was used in thebioreactor. In the Cellcube™, the crude virus product was harvested inthe form of virally infected cells, which is different from the virussupernatant harvested from the bioreactor.

Crude cell lysate (CCL), produced after 5 cycles freeze-thaw of theharvested virally infected cells, was purified by IEC using the abovedescribed method. Unlike the virus supernatant from the bioreactor, nosatisfactory purification was achieved for the CCL material generatedfrom the Cellcube™. FIG. 14 shows the chromatogram. The result suggeststhat crude virus solution generated from the Cellcube™ by freeze-thawingharvested cells is not readily purified by the IEC method.

Other purification methods, including hydrophobic interaction and metalchelate chromatography, were examined for the purification of virus inCCL. Unfortunately, no improvement in purification was observed byeither method. Considering the difficulties of purification of virus inCCL and the disadvantages associated with a freeze-thaw step in theproduction process, the inventors decided to explore other cell lysismethods.

A) Purification of Crude Virus Solution in Lysis Buffer

As described in Examples 1 and 3, HPLC analysis was used to screendifferent detergent lysis methods. Based on the HPLC results, 1%Tween-20 in 20 mM Tris+0.25 M NaCl+1 MM MgCl₂, pH=7.50 buffer wasemployed as the lysis buffer. At the end of the virus production phase,instead of harvesting the infected cells, the lysis buffer was pumpedinto the Cellcubem after draining the spent medium. Cells were lysed andvirus released into the lysis buffer by incubating for 30 min.

After clarification and filtration, the virus solution wasconcentrated/diafiltrated and treated with Benzonase to reduce thecontaminating nucleic acid concentration. The treated virus solution waspurified by the method developed above using Toyopearl SuperQ resin.Satisfactory separation, similar to that obtained using virussupernatant from the bioreactor, was achieved during elution. FIG. 15shows the elution profile. However, when the virus fraction was analyzedon HPLC, another peak in addition to the virus peak was detected. Theresult is shown in FIG. 16A.

To further purify the virus, the collected virus fraction wasre-purified using the same method. As shown in FIG. 16B, purity of thevirus fraction improved considerably after the second purification.Metal chelate chromatography was also evaluated as a candidate for thesecond purification. Similar improvement in virus purity as seen withthe second IEC was achieved. However, because of its simplicity, IEC ispreferred as the method of choice for the second purification.

As described above in Example 2, medium perfusion rate employed duringthe cell growth and virus production phases has a considerable impact onthe HPLC separation profile of the Tween-20 crude virus harvest. Forcrude virus solution produced under high medium perfusion rate, two ionexchange columns are required to achieve the required virus purity.

Based on the much improved separation observed on HPLC for virussolution produced under low medium perfusion rate, it is likely thatpurification through one ion exchange column may achieve the requiredvirus purity. FIG. 17 shows the elution profile using crude virussolution produced under low medium perfusion rate. A sharp virus peakwas attained during elution. HPLC analysis of the virus fractionindicates virus purity equivalent to that of CsCl gradient purifiedvirus after one ion exchange chromatography step. FIG. 18 shows the HPLCanalysis result.

The purified virus was further analyzed by SDS-PAGE, western blot forBSA, and nucleic acid slot blot to determine the contaminating nucleicacid concentration. The analysis results are given in FIG. 19A, FIG. 19Band FIG. 19C, respectively. All those analyses indicate that the columnpurified virus has equivalent purity compared to the double CsClgradient purified virus. Table 13 shows the virus titer and recoverybefore and after the column purification. For comparison purposes, thetypical virus recovery achieved by double CsCl gradient purification wasalso included. Similar virus recoveries were achieved by both methods.

TABLE 13 Comparison of IEC and double CsCl gradient ultracentrifugationpurification of AdCMVp53 from Cellcube ™ Titer (PFU/ml) A260/A280Particle/PFU Recovery IEC 1 × 10¹⁰ 1.27 36 63% Ultracentri- 2 × 10¹⁰1.26 38 60% fugation

A) Resin Capacity Study

The dynamic capacity of the Toyopearl Super Q resin was evaluated forthe purification of the Tween-20 harvested virus solution produced underlow medium perfusion rate. One hundred ml of resin was packed in a XK50column. Different amount of crude virus solution was purified throughthe column using the methods described herein.

Virus breakthrough and purification efficiency were analyzed on HPLC.FIG. 20 shows the HPLC analysis results. At a column loading factorgreater than sample/column volume ratio of 2:1, purity of the virusfraction was reduced. Contaminants co-eluted with the virus. At aloading factor of greater than 3:1, breakthrough of the virus into theflow through was observed. Therefore, it was proposed that the workingloading capacity of the resin be in the range of sample/column volumeratio of 1:1.

B) Concentration/Diafiltration Post Purification

A concentration/diafiltration step after column purification serves notonly to increase the virus titer, if necessary, but also to exchange tothe buffer system specified for the virus product. A 300K NMWC TFFmembrane was employed for the concentration step. Because of the absenceof proteinacious and nucleic acid contaminants in the purified virus,very high buffer flux was achieved without noticeable pressure dropacross the membrane.

Approximately 100% virus recovery was achieved during this step bychanging the buffer into 20 mM Tris+1 mM MgCl₂+0.15 M NaCl, pH=7.50. Thepurified virus was also successfully buffer exchanged into DPBS duringthe concentration/diafiltration step. The concentration factor can bedetermined by the virus titer that is desired in the final product andthe titer of virus solution eluted from the column. This flexibilitywill help to maintain the consistency of the final purified virusproduct.

C) Evaluation of Defective Adenovirus in the IEC Purified AdCMVp53

Due to the less than 100% packaging efficiency of adenovirus in producercells, some defective adenoviruses generally exist in crude virussolution. Defective viruses do not have DNA packaged inside the viralcapsid and therefore can be separated from intact virus on CsCl gradientultracentrifugation based the density difference. It is likely that itwould be difficult to separate the defective from the intact virusesbased on ion exchange chromatography assuming both viruses have similarsurface chemistry. The presence of excessive amount of defective viruseswill impact the quality of the purified product.

To evaluate the percentage of defective virus particles present, thepurified and concentrated viruses were subjected to isopicnic CsClultracentrifugation. As shown in FIG. 21, a faint band on top of theintact virus band was observed after centrifugation. Both bands wererecovered and dialyzed against 20 mM Tris+1 mM MgCl₂, pH=7.50 buffer toremove CsCl. The dialyzed viruses were analyzed on HPLC and the resultsare shown in FIG. 22. Both viruses show similar retention time. However,the defective virus has a smaller A260/A280 ratio than that of theintact virus. This is indicative of less viral DNA in the defectivevirus.

The peaks seen at retention times between 3.02 to 3.48 min are producedby glycerol which is added to the viruses (10% v/v) before freezing at−70° C. The percentage of the defective virus was less than 1% of thetotal virus. This low percentage of defective virus is unlikely toimpact the total particle to infectious virus (PFU) ratio in thepurified virus product. Both viruses were analyzed by SDS-PAGE (shown inFIG. 19A). Compared to the intact viruses, defective viruses lack theDNA associated core proteins banded at 24 and 48.5 KD. This result is inagreement with the absence of DNA in defective virus.

D) Process Overview of the Production and Purification of AdCMVpS3 Virus

Based on the above process development results, the inventors propose aproduction and purification flow chart for AdCMVp53 as shown in FIG. 23.The step and accumulative virus recovery is included with thecorresponding virus yield based on a 1 mer Cellcube™. The final virusrecovery is about 70±10%. This is about 3-fold higher than the virusrecovery reported by Huyghe et al. (1996) using a DEAE ion exchanger anda metal chelate chromatographic purification procedure for thepurification of p53 protein encoding adenovirus. Approximately 3×10¹²PFU of final purified virus product was produced from a 1 mer Cellcube™.This represents a similar final product yield compared to the currentproduction method using double CsCl gradient ultracentrifugation forpurification.

E) Scale-Up

Successful scale-up studies have been performed with the 4 mer Cellcube™system, and are currently underway to evaluate virus production in the16 mer Cellcube™ system. The crude virus solution produced will befiltered, concentrated and diafiltrated using a bigger Pelliconcassette. The quality and recovery of the virus will be determined.After Benzonase treatment, the crude virus solution will be purifiedusing a 20 cm and a 30 cm BioProcess column for the 4 mer and 16 mer,respectively.

Example 9

Improved Ad-p53 Production in Serum-Free Suspension Culture

Adaptation of 293 Cells

293 cells were adapted to a commercially available IS293 serum-freemedia (Irvine Scientific; Santa Ana, Calif.) by sequentially loweringdown the FBS concentration in T-flasks. The frozen cells in one vial ofPDWB were thawed and placed in 10% FBS DMEM media in T-75 flask and thecells were adapted to serum-free IS 293 media in T-flasks by loweringdown the FBS concentration in the media sequentially. After 6 passagesin T-75 flasks the FBS % was estimated to be about 0.019%. The cellswere subcultured two more times in the T flasks before they weretransferred to spinner flasks.

Serum-Free Adapted 293 Cells in T Flasks were Adapted to SuspensionCulture

The above serum-free adapted cells in T-flasks were transferred to aserum-free 250 mL spinner suspension culture (100 mL working volume) forthe suspension culture. The initial cell density was 1.18E+5 vc/mL.During the cell culture the viability decreased and the big clumps ofcells were observed. After 2 more passages in T-flasks the adaptation tosuspension culture was tried again. In a second attempt the media wassupplemented with heparin, at a concentration of 100 mg/L, to preventaggregation of cells and the initial cell density was increased to5.22E+5 vc/mL. During the cell culture there was some increase of celldensity and cell viability was maintained. Afterwards the cells weresubcultured in the spinner flasks for 7 more passages and during thepassages the doubling time of the cells was progressively reduced and atthe end of seven passages it was about 1.3 day which is comparable to1.2 day of the cells in 10% FBS media in the attached cell culture. Inthe serum-free IS 293 media supplemented with heparin almost all thecells existed as individual cells not forming aggregates of cells in thesuspension culture (Table 14).

TABLE 14 Serum-Free Suspension Culture: Adaptation to Suspension PassageNo. Flask No. Average Doubling Time (days) 11 Viability decreased 13 3.414 3.2 15 1 Viability decreased Heparin added 2 4.7 3 5.0 4 3.1 16 1 5.52 4.8 3 4.3 4 4.3 17 1 2.9 2 3.5 3 2.4 4 1.7 18 1 3.5 2 13.1 3 6.1 4 3.819 1 2.5 2 2.6 3 2.3 4 2.5 20 1 1.3 (97% viability) 2 1.5 (99%viability) 3 1.8 (92% viability) 4 1.3 (96% viability)Viral Production and Growth of Cells in Serum-Free Suspension Culture inSpinner Flask

To test the production of Ad5-CMVp53 vectors in the serum-freesuspension culture the above cells adapted to the serum-free suspensionculture were grown in 100 mL serum-free IS293 media supplemented with0.1% Pluronic F-68 and Heparin (100 mg/L) in 250mL spinner flasks. thecells were infected at 5 MOI when the cells reached 1.36E+06 viablecells/niL on day 3. The supernatant was analyzed everyday for HPLC viralparticles/mL after the infection. No viruses were detected other thanday 3 sample. On day 3 it was 2.2E+09 vps/mL. The pfu/mL on day 6 was2.6+/−0.6E+07 pfu/mL. The per cell pfu production was estimated to be 19which is approximately 46 times below the attached culture in theserum-supplemented media. As a control the growth of cells was checkedin the absence of an infection.

TABLE 15 Serum-Free Suspension Culture: Viral Production and Cell GrowthControl w/o Viral viral infection w/o Viral infection w/ infection mediaexchange media exchange Initial Density 2.1 × 10⁵ 2.1 × 10⁵ 2.1 × 10⁵(vc/mL) Cell Density at 9.1 × 10⁵ 1.4 × 10⁶ 1.5 × 10⁶ infection (vc/mL)Volumetric viral NA 2.6 × 10⁷ 2.8 × 10⁸ production (pfu/mL) 6 days P.I.Volumetric viral NA NA 1.3 × 10¹⁰ production (HPLC vps/mL) 6 days P.I.Per cell viral NA NA 1.3 × 10⁴ production (HPLC vps/cell)Preparation of Serum-Free Suspension Adapted 293 Cell Banks

As described above, after it was demonstrated the cells produce theAd-p53 vectors, the cells were propagated in the serum-free IS293 mediawith 0.1% F-68 and 100 mg/L heparin in the spinner flasks to makeserum-free suspension adapted cell banks which contain 1.0E+07 viablecells/mL/vial. To collect the cells they were centrifuged down when theywere at mid-log phase growth and the viability was over 90% andresuspended in the serum-free, supplemented IS293 media and centrifugeddown again to wash out the cells. Then the cells were resuspended againin the cryopreservation media which is cold IS293 with 0.1% F-68, 100mg/L heparin, 10% DMSO and 0.1% methylcellulose resulting in 1E+07viable cells/iL. The cell suspension was transferred to sterilecryopreservation vials and they were sealed and frozen in cryocontainerat −70 C overnight. The vials were transferred to liquid nitrogenstorage. The mycoplasma test was negative.

To revive the frozen cells one vial was thawed into the 50 mL serum-freeIS293 media with 0.1% F-68 and 100 mg/L heparin in a T-150. Since thenthe cultures were subcultured three times in 250 mL spinner flasks. Inthe other study one vial was thawed into 100 mL serum-free, supplementedIS293 media in a 250 mL spinner flask. Since then these were subculturedin serum-free spinner flasks 2 times. In both of the studies the cellsgrew very well.

Media Replacement and Viral Production in Serum-Free Suspension Culturein Spinner Flask

In the previous serum-free viral production in the suspension culture inthe spinner flask the per cell viral production was too low for theserum-free suspension production to be practical. It was supposed thatthis might be due to the depletion of nutrients and/or the production ofinhibitory byproducts. To replace the spent media with fresh serum-free,supplemented IS293 media the cells were centrifuged down on day 3 andresuspended in a fresh serum-free IS-293 medium supplemented with F-68and heparin (100 mg/L) and the resulting cell density was 1.20E+06 vc/mLand the cells were infected with Ad5-CMVp53 vectors at 5 MOI. Theextracellular HPLC vps/mL was 7.7E+09 vps/mL on day 3, 1.18E+10 vps/mLon day 4, 1.2E+10 vps/mL on day 5 and 1.3E+10 vps/mL on day 6 and thepfu/mL on day 6 was 2.75+/−0.86E+08 tvps/mL. The ratio of HPLC viralparticles to pfus was about 47. Also the cells have been centrifugeddown and lysed with the same type of the detergent lysis buffer as usedin the harvest of CellCube. The cellular HPLC vps/mL was 1.6E+10 vps/mLon day 2, 6.8E+09 vps/mL on day 3, 2.2E+09 vps/mL on day 4, 2.24E+09vps/mL on day 5 and 2.24E+09 vps/mL on day 6.

The replacement of the spent media with a fresh serum-free, supplementedIS 293 media resulted in the significant increase in the production ofAd-p53 vectors. The media replacement increased the production ofextracellular HPLC viral particles 3.6 times higher above the previouslevel on day 3 and the production of extracellular pfu titer ten timeshigher above the previous level on day 6. Per cell production of Ad-p53vectors was estimated to be approximately 1.33E+04 HPLC vps.

The intracellular HPLC viral particles peaked on day 2 following theinfection and then the particle numbers decreased. In return theextracellular viral particles increased progressively to the day 6 ofharvest. Almost all the Ad-p53 vectors were produced for the 2 daysfollowing the infection and intracellularly localized and then theviruses were released outside of the cells. Almost half of the viruseswere released outside of the cells into the supernatant between day 2and day 3 following the infection and the rate of release decreased astime goes on.

All the cells infected with Ad-p53 vectors lost their viability at theend of 6 days after the infection while the cells in the absence ofinfection was 97% viable. In the presence of infection the pH of thespent media without the media exchange and with the media exchange was6.04 and 5.97, respectively, while the one in the absence of theinfection was 7.00 (Table 14).

Viral Production and Cell Culture in Stirred Bioreactor with MediaReplacement and Gas Overlay

To increase the production of Ad-p53 vectors, a 5L CelliGen bioreactorwas used to provide a more controlled environment. In the 5 L CelliGenbioreactor the pH and the dissolved oxygen as well as the temperaturewere controlled. Oxygen and carbon dioxide gas was connected to thesolenoid valve for oxygen supply and the pH adjustment, respectively.For a better mixing while generating low shear environment amarine-blade impeller was implemented. Air was supplied all the timeduring the operation to keep a positive pressure inside the bioreactor.

To inoculate the bioreactor a vial of cells was thawed into 100 mLserum-free media in a 250 mL spinner flask and the cells were expandedin 250 or 500 mL spinner flasks. 800 mL cell inoculum, grown in 500 mLflasks, was mixed with 2700 mL fresh media in a 10 L carboy andtransferred to the CelliGen bioreactor by gas pressure. The initialworking volume of the CelliGen bioreactor was about 3.5 L culture. Theagitation speed of the marine-blade impeller was set at 80 rpm, thetemperature at 37° C., pH at 7.1 at the beginning and 7.0 after theinfection and the DO at 40% all the time during the run.

The initial cell density was 4.3E+5 vc/mL (97% viability) and 4 dayslater when the cell density reached to 2.7E+6 vc/mL (93% viability) thecells were centrifuged down and the cells were resuspended in a freshmedia and transferred to the CelliGen bioreactor. After the mediaexchange the cell density was 2.1E+6 vc/mL and the cells were infectedat MOI of 10. Since then the DO dropped to below 40%. To keep the DOabove 40%, about 500 mL of culture was withdrawn from the CelliGenbioreactor to lower down the oxygen demand by the cell culture and theupper marine-blade was positioned close to the interface between the gasand the liquid phase to improve the oxygen transfer by increasing thesurface renewal. Since then the DO could be maintained above 40% untilthe end of the run.

For pH control, CO₂ gas was used to acidify the cell culture and 1 NNaHCO₃ solution to make the cell culture alkaline. The pH control wasinitially set at 7.10. The initial pH of the cell culture was about pH7.41. Approximately 280 mL 1N NaHCO₃ solution was consumed until the pHof cell culture stabilized around pH 7.1. After the viral infection ofthe cell culture, the pH control was lowered down to pH 7.0 and the CO₂gas supply line was closed off to reduce the consumption of NaHCO₃solution. The consumption of too much NaHCO₃ solution for pH adjustmentwould increase the cell culture volume undesirably. Since then 70 mL 1NNaHCO₃ solution was consumed and the pH was in the range between 7.0 and7.1 most of the time during the run. The temperature was controlledbetween 35° C. and 37° C.

After the infection the viability of the cells decreased steadily untilday 6 of harvest after the infection. On the harvest day none of thecells was viable. The volumetric viral production of the CelliGenbioreactor was 5.1E+10 HPLC vps/mL compared to the 1.3E+10 vps/mL in thespinner flask. The controlled environment in the CelliGen bioreactorincreased the production of Ad-p53 vectors 4-fold compared to thespinner flasks with media replacement. This is both due to the increaseof the cell density at the time of infection from 1.2E+6 to 2.1E+6 vc/mLand the increase of per cell viral production from 1.3E+4 to 2.5E +4vps/mL. The 2.5E+4 vps/mL is comparable to the 3.5E+4 vps/cell in theserum-supplemented, attached cell culture.

Viral Production and Cell Culture in Stirred and Sparged Bioreactor

In the first study the cells were successfully grown in an stirredbioreactor for viral production, and the oxygen and CO₂ were supplied bygas overlay in the headspace of a bioreactor. However, this method willlimit the scale-up of the cell culture system because of its inefficientgas transfer. Therefore in the second study, to test the feasibility ofthe scale up of the serum-free suspension culture was investigated bygrowing of cells and producing Ad-p53 in a sparged bioreactor. Pureoxygen and CO₂ gases were supplied by bubbling through the serum-freeIS293 media supplemented with F-68 (0.1%) and heparin (100 mg/L).

Pure oxygen was bubbled through the liquid media to supply the dissolvedoxygen to the cells and the supply of pure oxygen was controlled by asolenoid valve to keep the dissolved oxygen above 40%. For efficientoxygen supply while minimizing the damage to the cells, a stainlesssteel sintered air diffuser, with a nominal pore size of approximately0.22 micrometer, was used for the pure oxygen delivery. The CO₂ gas wasalso supplied to the liquid media by bubbling from the same diffuser asthe pure oxygen to maintain the pH around 7.0. For pH control, Na₂CO₃solution (106 g/L) was also hooked up to the bioreactor. Air wassupplied to the head space of the bioreactor to keep a positive pressureinside the bioreactor. Other bioreactor configuration was the same asthe first study.

Inoculum cells were developed from a frozen vial. One vial of frozencells (1.0E+7 vc) was thawed into 50 mL media in a T-150 flask andsubcultured 3 times in 200 mL media in 500 mL spinner flasks. 400 L ofinoculum cells grown in 2 of 500 mL spinner flasks were mixed with IS293media with F-68 and heparin in a 10 L carboy to make 3.5 L cellsuspension and it was transferred to the 5 L CelliGen bioreactor.

The initial cell density in the bioreactor was 3.0E+4 vc/mL. The initialcell density is lower than the first study. In the first study four of500 mL spinner flasks were used as the inoculum. Even with the lowerinitial cell density the cells grew up to 1.8E+6 vc/mL on day 7 in thesparged environment and the viability was 98%. During the 7 days'growth, glucose concentration decreased from 5.4 g/L to 3.0 g/L andlactate increased from 0.3 g/L to 1.8 g/L.

On day 7, when the cell density reached 1.8E+6 vc/mL, the cells in thebioreactor were centrifuged down and resuspended in 3.5 L freshserum-free IS293 media with F-68 and heparin in a 10 L carboy. The 293cells were infected with 1.25E+11 pfu Ad-p53 and transferred to theCelliGen bioreactor. In the bioreactor, cell viability was 100% but thecell density was only 7.2E+5 vc/mL. There was a loss of cells during themedia exchange operation. The viral titer in the media was measured as2.5E+10 HPLC vps/mL on day 2, 2.0E+10 on day 3, 2.8E+10 on day 4,3.5E+10 on day 5 and 3.9E+10 HPLC vps/mL on day 6 of harvest. The firstCelliGen bioreactor study with gas overlay produced 5.1E+10 HPLC vps/mL.The lower virus concentration in the second run was likely due to thelower cell density at the time of infection. Compared to the 7.2E+5vc/mL in the second run, 2.1E+6 vc/mL was used in the first run.Actually the per cell production of Ad-p53 in the second spargedCelliGen bioreactor is estimated to be 5.4E+4 vps/cell which is thehighest per cell production ever achieved so far. The per cellproduction in the first serum-free CellGen bioreactor without spargingand the serum-supplemented T-flask was 2.5E+4 vps/cell and 3.5E+4vps/cell, respectively.

After the viral infection, the viability of the cells decreased from100% to 13% on day 6 of harvest. During those 6 days after the infectionthe glucose concentration decreased from 5.0 g/L to 2.1 g/L and thelactate increased from 0.3 g/L to 2.9 g/L. During the entire period ofoperation about 20 mL of Na₂CO₃ (106 g/L) solution was consumed.

The experimental result shows that it is technically and economicallyfeasible to produce Ad-p53 in the sparged and stirred bioreactor.Scale-up and large-scale unit operation of sparged and stirredbioreactor are well established.

Example 10 Blanche et al Production Process

The following example is text excerpted from pages 4–14 of Blanche et alin U.S. Ser. No. 60/076,662. This text is descriptive of the methodsused by Blanche et al in production of recombinant adenovirus.

Recombinant adenoviruses are usually produced by the introduction ofviral DNA into the encapsulation line, followed by lysis of the cellsafter approximately 2 or 3 days (with the kinetics of the adenoviralcycle being 24 to 36 hours). After lysis of the cells, the recombinantviral particles are isolated by centrifugation on a cesium chloridegradient.

For implementation of the process, the viral DNA introduced may be thecomplete recombinant viral genome, possibly constructed in a bacterium(ST 95010) or in a yeast (WO95/03400), transfected in the cells. It mayalso be a recombinant virus used to infect the encapsulation line. It isfurther possible to introduce the viral DNA in the form of fragments,each carrying a portion of the recombinant viral genome and a homologyzone permitting the recombinant viral genome to be reconstituted byhomologous recombination between the different fragments afterintroduction into the encapsulation cell. Thus a classical adenovirusproduction process includes the following steps: The cells (for example,cells 293) are infected in a culture plate with a viral prestock at therate of 3 to 5 viral particles per cell (Multiplicity of Infection(MOI)=3 to 5), or transfected with viral DNA. The incubation then lasts40 to 72 hours. The virus is subsequently released from the nucleus bylysis of the cells, generally by several successive thaw cycles. Thecellular lysate obtained is then centrifuged at low speed (2000 to 4000rpm), after which the supernatant (clarified cellular lysate) ispurified by centrifugation in the presence of cesium chloride in twosteps:

A first rapid 1.5 hour centrifugation on two layers of cesium chlorideof densities 1.25 and 1.40 surrounding the density of the virus (1.34)in such a way as to separate the virus from the proteins of the medium;

A second, longer centrifugation in a gradient (from 10 to 40 hoursaccording to the rotor used), which constitutes the true and onlypurification step of the virus.

Generally, after the second centrifugation step, the band of the virusis intensified. Nevertheless, two finer, less dense bands are observed.Observation under the electron microscope has shown that these bands aremade up of empty or broken viral particles for the denser band and ofviral subunits (pentons, hexons) for the less dense band. After thisstep, the virus is harvested by needle puncture in the centrifugationtube and the cesium is eliminated by dialysis or deionization.

Although the purity levels obtained are satisfactory, this type ofprocess presents certain drawbacks. In particular, it is based on theuse of cesium chloride, which is a reagent incompatible with therapeuticuse in man. Thus, it is imperative to eliminate the cesium chloride atthe end of purification. This process also has certain otherdisadvantages mentioned below, limiting its use to an industrial scale.

To remedy these problems, it has been proposed to purify the virusobtained after lysis, not by gradient of cesium chloride, but bychromatography. Thus the article of Huyghe et at. (Hum. Gen. Ther. 6(1996) 1403) describes a study of different types of chromatographiesapplied to the purification of recombinant adenoviruses. This articledescribes in particular a study of recombinant adenovirus purificationusing weak anion exchange chromatography (DEAE). Earlier studies alreadydescribed the use of this type of chromatography toward that goal(Klemperer et al., Virology 9 (1959) 536; Philipson, L., Virology 10(1960) 459; Haruna et al., Virology 13 (1961) 264). The resultspresented in the article by Huyghe et al. show a rather poor efficacy ofthe ion exchange chromatography protocol recommended. Thus, theresolution obtained is average, with the authors indicating that virusparticles are present in several chromatographic peaks; the yield is low(viral particle yield: 67%; infectious particle yield: 49%); and theviral preparation obtained following this chromatographic step isimpure. In addition, pretreatment of the virus with differentenzymes/proteins is necessary. This same article also describes a studyof the use of gel permeation chromatography, showing very poorresolution and very low yields (15–20%).

The present invention describes a new process for the production ofrecombinant adenoviruses. The process according to the invention resultsfrom changes in previous processes in the production phase and/or in thepurification phase. The process according to the invention now makes itpossible in a very rapid and industrializable manner to obtain stocks ofvirus of very high quantity and quality.

One of the first features of the invention concerns more particularly aprocess for the preparation of recombinant adenoviruses in which theviruses are harvested from the culture supernatant. Another aspect ofthe invention concerns a process for the preparation of adenovirusesincluding an ultrafiltration step. According to yet another aspect, theinvention concerns a process for the purification of recombinantadenoviruses including an anion exchange chromatography step. Thepresent invention also describes an improved purification process, usinggel permeation chromatography, possibly coupled with anion exchangechromatography. The process according to the invention makes it possibleto obtain viruses of high quality in terms of purity, stability,morphology, and infectivity, with very high yields and under productionconditions completely compatible with the industrial requirements andwith the regulations concerning the production of therapeutic molecules.

In particular, in terms of industrialization, the process according tothe invention uses methods of the treatment of supernatants of culturestested on a large scale for recombinant proteins, such asmicrofiltration or deep filtration, and tangential ultrafiltration.Furthermore, because of the stability of the virus at 37° C., thisprocess permits better organization at the industrial stage inasmuch as,contrary to the intracellular method, the harvesting time does not needto be precise to within a half day. Moreover, it guarantees maximumharvesting of the virus, which is particularly important in the case ofviruses defective in several regions. In addition, the process accordingto the invention permits an easier and more precise follow-up of theproduction kinetics directly on homogenous samples of supernatant,without pretreatment, which permits better reproducibility of theproductions. The process according to the invention also makes itpossible to eliminate the cell lysis step. The lysis of the cellspresents a number of drawbacks. Thus, it may be difficult to considerbreaking the cells by freeze/thaw cycles at the industrial level.Besides, the alternative lysis methods (Dounce, X-press, sonification,mechanical shearing, etc.) present drawbacks as well: they are potentialgenerators of sprays that are difficult to confine for L2 or L3 viruses(level of confinement of the viruses, depending on their pathogenicityof their mode of dissemination), with these viruses having a tendency tobe infectious through airborne means; they generate shear forces and/ora liberation of heat that are difficult to control, diminishing theactivity of the preparations. The solution of using detergents to lysethe cells would demand validation and would also require thatelimination of the detergent be validated. Finally, cellular lysis leadsto the presence in the medium of a large quantity of cellular debris,which makes purification more difficult. In terms of virus quality, theprocess according to the invention potentially permits better maturationof the virus, leading to a more homogenous population. In particular,provided that the packing of the viral DNA is the last step in the viralcycle, the premature lysis of the cells potentially liberates emptyparticles which, although not replicative, are a priori infectious andcapable of participating in the distinctive toxic effect of the virusand of increasing the ratio of specific activity of the preparationsobtained. The ratio of specific infectivity of a preparation is definedas the ratio of the total number of viral particles, measured bybiochemical methods (OD 260 nm, HPLC, CRP, immuno-enzymatic methods,etc.), to the number of viral particles generating a biologic effect(formation of lysis plaques on cells in culture and solid medium,translation of cells). In practice, for a purified preparation, thisratio is determined by dividing the concentration of particles measuredby OD at 260 nm by the concentration of plaque-forming units in thepreparation. This ratio should be less than 100.

The results obtained show that the process according to the inventionmakes it possible to obtain a virus of a purity comparable to thehomologous one purified by centrifugation in cesium chloride gradient,in a single step and without preliminary treatment, starting from aconcentrated viral supernatant.

A first goal of the invention thus concerns a process for the productionof recombinant adenoviruses characterized by the fact that the viral DNAis introduced into a culture of encapsulation cells and the virusesproduced are harvested after release into the culture supernatant.Contrary to the previous processes in which the viruses are harvestedfollowing premature cellular lysis performed mechanically or chemically,in the process according to the invention the cells are not lysed bymeans of an external factor. Culturing is pursued during a longer periodof time, and the viruses are harvested directly in the supernatant,after spontaneous release by the encapsulation cells. In this way thevirus according to the invention is recovered in the cellularsupernatant, while in the previous processes it is an intracellular andmore particularly an intranuclear virus that is involved.

The applicant has now shown that despite that elongation in duration ofthe culture and despite the use of larger volumes, the process accordingto the invention makes it possible to generate viral particles in largequantity and of better quality.

In addition, as indicated above, this process makes it possible to avoidthe lysis steps, which are cumbersome from the industrial standpoint andgenerate numerous impurities.

The principle of the process thus lies in the harvesting of the virusesreleased into the supernatant. This process may involve a culture timelonger than that used in the previous techniques based on lysis of thecells. As indicated above, the harvesting time does not have to beprecise to within a half day. It is essentially determined by thekinetics of release of the viruses into the culture supernatant.

The kinetics of liberation of the viruses can be followed in differentways. In particular, it is possible to use analysis methods such asreverse-phase HPLC, ion exchange analytic chromatography,semiquantitative PCR (example 4.3), staining of dead cells with trypanblue, measurement of liberation of LDH type intracellular enzymes,measurement of particles in the supernatant by Coulter type equipment orby light diffraction, immunologic (ELISA, RIA, etc.) or nephelometricmethods, titration by aggregation in the presence of antibodies, etc.

Harvesting is preferably performed when at least 50% of the viruses havebeen released into the supernatant. The point in time at which 50% ofthe viruses have been released can easily be determined by doing akinetic study according to the methods described above. Even morepreferably, harvesting is performed when at least 70% of the viruseshave been released into the supernatant. It is particularly preferred todo the harvesting when at least 90% of the viruses have been releasedinto the supernatant, i.e., when the kinetics reach a plateau. Thekinetics of liberation of the virus are essentially based on thereplication cycle of the adenovirus and can be influenced by certainfactors. In particular, they may vary according to the type of virusused, and especially according to the type of deletion done in therecombinant viral genome. In particular, deletion of region E3 seems toslow liberation of the virus. Thus, in the presence of region E3, thevirus can be harvested between 24 and 48 hours post-infection. Incontrast, in the absence of region E3, a longer culturing time seemsnecessary. In this regard, the applicant has had experience with thekinetics of liberation of an adenovirus deficient in regions E1 and E3into the supernatant of the cells, and has shown that liberation beginsapproximately 4 to 5 days post-infection and lasts up to about day 14.Liberation generally reaches a plateau between day 8 and day 14, and thetiter remains stable for at least 20 days post-infection.

Preferably, in the process according to the invention, the cells arecultured during a period ranging between 2 and 14 days. Furthermore,liberation of the virus may be induced by expression in theencapsulation cell of a protein, for example a viral one, involved inthe liberation of the virus. Thus, in the case of the adenovirus,liberation may be modulated by expression of the Death protein coded byregion E3 of the adenovirus (protein E3-11.6K), possibly expressed underthe control of an inducible promoter. Consequently, it is possible toreduce the virus liberation time and to harvest in the culturesupernatant more than 50% of the viruses 24–48 hours post-infection.

To recover the viral particles, the culture supernatant isadvantageously first filtered. Since the adenovirus is approximately 0.1μm (120 nm) in size, filtration is performed with membranes whose poresare sufficiently large to let the virus pass through, but sufficientlyfine to retain the contaminants. Preferably, filtration is performedwith membranes having a porosity greater than 0.2 μm. According to aparticularly advantageous exemplified embodiment, filtration isperformed by successive filtrations on membranes of decreasing porosity.Particularly good results have been obtained by doing filtration onfilters with a range of decreasing porosity—10 μm, 1.0 μm, then 0.8–0.2μm. According to another preferred variant, filtration is performed bytangential microfiltration on flat membranes or hollow fibers. Moreparticularly, it is possible to use flat Millipore membranes or hollowfibers ranging in porosity between 0.2 and 0.6 μm. The results presentedin the examples show that this filtration step has a yield of 100% (noloss of virus was observed by retention on the filter having the lowestporosity).

According to another aspect of the invention, the applicant has nowdeveloped a process making it possible to harvest and purify the virusfrom the supernatant. Toward this goal, a supernatant thus filtered (orclarified) is subjected to ultrafiltration. This ultrafiltration makesis possible (i) to concentrate the supernatant, with the volumes usedbeing important; (ii) to do a first purification of the virus and (iii)to adjust the buffer of the preparation in the subsequent preparationsteps. According to a preferred exemplified embodiment, the supernatantis subjected to tangential ultrafiltration. Tangential ultrafiltrationconsists of concentrating and fractionating a solution between twocompartments, retentate and filtrate, separated by membranes ofspecified cutoff thresholds, by producing a flow in the retentatecompartment of the apparatus and by applying a transmembrane pressurebetween this compartment and the filtrate compartment. The flow isgenerally produced with a pump in the retentate compartment of theapparatus, and the transmembrane pressure is controlled by a valve onthe liquid channel of the retentate circuit or by a variable-speed pumpon the liquid channel of the filtrate circuit. The speed of the flow andthe transmembrane pressure are chosen so as to generate low shear forces(Reynolds number less than 5000 sec⁻¹, preferably below 3000 sec⁻¹,pressure below 1.0 bar), while preventing plugging of the membranes.Different systems can be used to accomplish ultrafiltration, e.g.,spiral membranes (Millipore, Amicon), as well as flat membranes orhollow fibers (Amicon, Millipore, Sartorius, Pall, GF, and Sepracor).Since the adenovirus has a mass of ca. 1000 kDa, it is advantageouswithin the scope of the invention to use membranes having a cutoffthresh below 1000 kDa, and preferably ranging between 100 kDa and 1000kDa. The use of membranes having a cutoff threshold of 1000 kDa orhigher in effect causes a large loss of virus at this stage. It ispreferable to use membranes having a cutoff threshold ranging between200 and 600 kDa, and even more preferable, between 300 and 500 kDa. Theexperiences presented in the examples show that the use of a membranehaving a cutoff threshold at 300 kDa permits more than 90% of the viralparticles to be retained, while eliminating the contaminants from themedium (DNA, proteins in the medium, cellular proteins, etc.). The useof a cutoff threshold of 500 kDa offers the same advantages.

The results presented in the examples show that this step makes itpossible to concentrate large volumes of supernatant without loss ofvirus (90% yield), with generation of a better quality virus. Inparticular, concentration factors of 20- to 100-fold can easily beobtained.

This ultrafiltration step thus includes an additional purificationcompared to the classical model inasmuch as the contaminants of massbelow the cutoff threshold (300 or 500 kDa) are eliminated at least inpart. A distinct improvement in the quality of the viral preparation maybe seen upon comparing the appearance of the separation after the firstultracentrifugation step according to the two processes. In theclassical process involving lysis, the viral preparation tube presents acloudy appearance with a coagulum (lipids, proteins) sometimes touchingthe virus band, while in the process according to the invention,following liberation and ultrafiltration, the preparation presents aband that is already well resolved of the contaminants of the mediumthat persist in the upper phase. An improvement in quality is alsodemonstrated upon comparing the profiles on ion exchange chromatographyof a virus obtained by cellular lysis with a virus obtained byultrafiltration as described in the present invention. In addition, itis possible to further enhance the quality by pursuing ultrafiltrationwith diafiltration of the concentrate. This diafiltration is performedbased on the same principle as tangential ultrafiltration, and makes itpossible to more completely eliminate the large-sized contaminants atthe cutoff threshold of the membrane, while achieving equilibration ofthe concentrate in the purification buffer.

In addition, the applicant has also shown that this ultrafiltrationmakes it possible to purify the virus directly by ion exchangechromatography or by gel permeation chromatography, permitting excellentresolution of the viral particle peak without requiring treatment of thepreparation beforehand with chromatography. This is particularlyunexpected and advantageous. In fact, as indicated in the article byHuyghe et al. mentioned above, purification by chromatography of viralpreparations gives poor results and also requires pretreatment of theviral suspension with benzonase and cyclodextrins.

Example 11 Optimization of Production Process

To arrive at an optimized process that may be used for adenovirusproduction for clinical therapeutic production, a few steps in the aboveprocess as well as that of Blanche et al in PCT Publication No. WO98/00524 (incorporated by reference) have been modified to enhance largescale production. Those steps involve modification to the virus harveststep, the nuclease treatment step, and the resin used for purification.The optimized process is depicted by the flow chart in FIG. 28.

Virus Harvest Step

In the process described above, virus was harvested by lysing the 293cells using a 1% Tween-20 lysis solution 2 days post-viral infection.This harvest method required the introduction of a lysis step into theprocess and the addition of one substance (Tween-20) into the crudeviral harvest. In consideration of the lytic nature of the adenoviruslife cycle, an alternative strategy was used to harvest thevirus-containing supernatant after complete viral-mediated 293 celllysis. Viral release kinetics were determined by analyzing daily samplesof supernatant from the CellCube™ system after infection. Viral releaseinto the supernatant reached a plateau on day 5 post-infection. Thekinetics of viral release were found to be consistent. FIG. 24 shows thetypical viral release kinetics for Ad5CMV-p53. Equivalent viral yieldwas obtained by using either the Tween-20 lysis or the autolysissupernatant harvest methods. The supernatant harvest method, however,simplified the production process by removing the lysis step in theprocess and the added lysis agent (Tween-20) in the crude viral harvest.As a result, the supernatant harvest method will preferably be used forthe optimized process.

Nuclease Treatment Step

In the above process and that of Blanch et al in PCT Publication No. WO98/00524, 1M NaCl was included in the Benzonase™ treatment buffer toprevent viral precipitation during enzyme treatment. Unfortunately, thepresence of 1M NaCl in the buffer was found to significantly inhibit theBenzonase™ enzymatic activity. As a result, other buffers which couldprevent viral precipitation without retarding the Benzonase™ enzymaticactivity were examined. A 0.5M Tris/HCl+1 mM MgCl₂, pH=8.0, buffer wasfound to meet both criteria. In addition, this buffer has a conductivityof 19 mS/cm, which makes it possible to load the Benzonase™-treatedviral solution directly onto the chromatographic column for purificationAs a result, changing to the 0.5M Tris/HCl+1 mM MgCl₂, pH=8.0, bufferwill not only improve the Benzonase™ treatment efficiency but alsosimplify the downstream process.

Resin for Purification

The Fractogel TMAE(s) resin and Toyopearl SuperQ 650M resin employed inthe above process and that of Blanche et al performed consistently well.However because of supply and technical support problems, alternativeresins were chosen for use in virus purification. Source 15Q resinmanufactured by Pharmacia Biotech was found to perform as well as theFractogel and Toyopearl resins. FIG. 25 shows a typical chromatogramfrom the Source 15Q resin. Suprisingly, viral material was found tointeract slightly stronger with the Source 15Q resin than with theFractogel and Toyopearl resin. As a result, a larger viral protein peakwas seen at the beginning of the gradient elution. The purified virusfraction was also eluted relatively later in the gradient. However, theoverall purification profile was not significantly different from thatof the Fractogel or Toyopearl resin. HPLC analysis of the purified viralfraction from the Source 15Q resin showed an equivalent profile to thatfrom the Fractogel resin. FIG. 26 shows the HPLC profile.

Ad5CMV-p53 made by the optimized process was also assessed forbiological activity compared to material made by the above process andthat of Blanche et al. Two cell lines, H1299 and SAOS-LM, which expressno endogenous p53, were transduced with materials made by the twoprocesses at equal multiplicities of infection (viral particles/cell).p53 expression was monitored at 6 hours post-transduction in H1299 and24 hours post-transduction in SAOS-2. The level of p53 expressionmediated by the two materials was equivalent and dose-dependent in bothrecipient cell lines (FIG. 27).

Process Hold Points

The freeze and thaw stability of the purified viral fraction eluted fromthe chromatography column (purified bulk) was evaluated. The purifiedviral fraction eluted from the column was frozen bulk at ≦−60° C. aftersupplementing with glycerol to a final concentration of 10% (v/v). Thefrozen bulk was thawed successfully without detrimental effects ontiter. The freeze and thaw data are given in Table 16.

TABLE 16 Freeze and thaw of purified bulk Post freeze-thaw Small volumeNo freeze Bulk freeze (45 ml) freeze (1 ml) Viral particles/ml 4.0 ×10¹¹ 3.8 × 10¹¹ 4.1 × 10¹¹

Furthermore, no change in HPLC profiles was observed pre- andpost-freeze and thaw. Therefore, viral material at thepost-chromatography step can be held at <−60° C. for further processing,and a process hold can be introduced at the post-chromatography step(purified bulk).

Similar freeze and thaw stability was observed for formulated sterilebulk product. Table 17 shows the freeze and thaw data.

TABLE 17 Freeze and thaw of formulated sterile bulk product Postfreeze-thaw Small volume freeze No freeze Bulk freeze (45 ml) (1 ml)Viral particles/ml 1.3 × 10³ 1.4 × 10¹³ 1.3 × 10¹³

As a result, the formulated sterile bulk product can be held at <−60° C.before aseptic filling without damaging effects on the viral titer and aprocess hold point can be introduced at the post-formulation step(sterile bulk).

Example 12 Parameters for Large Scale Production

During the scale up and optimization of the large scale process(16-mer), several parameters were found by the inventors to be desirablefor successful production runs and high virus yield. These desirableparameters are centered around the cell culturing system, the mostupstream portion of the adenovirus production process, and are believedto be applicable to other types of cell culturing systems and at largerscales. In particular, it can be easily envisioned that the changesdescribed below which result in finctional changes to the system will beuseful to enable modification and optimization of other cell culturesystems.

For the present example, the culture control parameters are as follows.Cells are cultured at 37° C. with 10% CO₂. Cell culture medium isDMEM+10% FBS, and the inoculation cell density for cell expansion is<4×10⁴ cells/cm². The parameters that involve the set up and executionof the CellCube™ system and are listed below.

CellCube™ Setup: In the full scale set up (4×100 or “16-mer”), it isdesirable to use one separate cell culture medium recirculation loop foreach cube module (4-mer) to achieve even medium perfusion. For example,in the present 16-mer set-up, the 16-mer is composed of four 4-merslinked together in a series, each 4-mer having it's own mediumreciruclation loop. The 16-mer is considered one unit and is controlledby a single control module that modulates the rate of medium perfusionand measures the culture control parameters. Other setups such as usingone medium recirculation loop for every two 4-mer modules results inuneven medium perfusion due to pressure drops in the system, and isdetrimental to the health of the cells in the second cube with lowerlevels of nutrients and freshly oxygenated medium. Thus, in a cellculture system used for adenovirus production, it is preferable that thecell culture medium perfusion be maintained at a constant pressure andrate, ensuring consistent and optimal health of the producer cells. Theperfusion rate is determined by monitoring one or more of the cellculture control parameters, such as glucose concentration.

Seeding Density: In order to achieve maximal cell expansion and growth,it is most preferable to inoculate the CellCube™ with 1–2×10⁴ cells/cm².Higher numbers of cells used in the cell inoculation step results in acell density that is too high and the result is an over-confluence ofcells at the time of viral infection, thus lowering yields. It is wellwithin one of skill in the art to determine that in other types of cellculturing systems, similar optimization of the seeding density for aparticular system could easily be determined.

Seeding Method: It has been found that for full scale production, it isadvantageous to use one homogeneous cell pool for seeding of allCellCube™ modules. Prior to seeding the cell culture apparatus, producercells from the working cell bank are expanded from stock cultures. Thiscell expansion is accomplished by growing the cells in tissue cultureflasks or other similar cell culture devices, and continual splitting ofthe cells into larger tissue culture devices. Upon reaching the totalnumber of needed cells for inoculation of the large scale cell cultureapparatus, all of the cells from each of the cell culture devices usedfor cell expansion are pooled together. This homogeneous cell pool isused to inoculate each of the CellCube™ modules of the 16-mer. Seedingof each of the modules using separate cell populations, for example fromindividual cell culture devices used in the cell expansion phase, canresult in uneven cell density, and therefore uneven confluency levels atthe time of infection. It is believed that the use of a homogeneous cellpool for seeding overcomes these problems.

Length of Cell Inoculation: During inoculation of each of the CellCube™modules, cells are added to the module and allowed a period of a fewhours to attach to the surface of the module. During this time there isno medium perfusion or recirculation. It has been found by the inventorsthat it is advantageous to complete this cell seeding in one day (24hrs). Thus for example, one side of the module is inoculated and leftfor a period of 6–8 hours to allow cell attachment, and then the otherside of the module is inoculated and leftovernight to allow the cells toattach to the surface of the module. During this seeding process, thecell culture medium from each side of the module is kept separate, andnot allowed to flow to the other side of the module. It has beenobserved that if the cell inoculation procedure is done over a period oftime longer than one day, and/or with medium exchange between sides ofthe module, that there is a greater likelihood of cell detachment fromthe cell culture surface due to weak attachment. Possible reasons forthis weak attachment may include: 1) the medium exchange between sidesof the module which may produce shear forces with the potential todislodge cells undergoing the attachment process, and 2) the longer timeperiod before medium perfusion is started may result in low levels ofnutrients in the media, and therefore the health of the cellsdeteriorates, leading to less efficient attachment.

Culture Control Parameters: The inventors have found that glucoseconcentration of the cell culture medium should preferably be maintainedat 1–2 g/L. Previous studies using glucose concentrations at higherlevels has been shown to reduce product yield.

Infection Method: Eight days post-cell seeding, the cells are infectedwith adenovirus. During the infection process, medium perfusion isstopped for one hour, however medium recirculation is maintained, thuskeeping high levels of fresh oxygen in the medium. It has been found bythe inventors that if medium recirculation is also stopped during theinfection step, there is an increased possibility of cell death due tooxygen starvation.

Example 13 Optimized Large Scale Production and Purification ofAdenovirus

The example described below is descriptive of the methods and materialsused in a large scale production and purification process forrecombinant Ad5CMV-p53 adenovirus. This process uses a CellCube™bioreactor apparatus as the cell culturing system, and large scale inthis example refers to a CellCube 4×100 set up or multiples thereof.Total maximum virus yields that may be obtained from one CellCube 4×100system are about 1–5×10¹⁵ viral particles at harvest.

Cell Expansion and Culture

The CellCube™ 4×100 was set up as described above, with 4 CellCube™ 100modules in parallel, all in a medium recirculation loop, and the wholesystem being controlled by a single control unit. The producer cells,293 cells from a working cell bank (WCB), were thawed and expanded in Tflasks and Cellfactories (Nunc) seeding at densities from 1–8×10e4cells/cm². Cells were generally split at a confluence of about 85–90%and continually expanded until enough cells were obtained forinoculation of the Cellcube™. At the end of the cell expansion phase,all the cells from each of the Cellfactories were pooled to make onehomogeneous mixture of 293 cells. This cell pool was used to inoculatethe Cellcubem at a total cell number in the range of 1–3×10e9 viablecells per side. During cell inoculation, medium perfusion andrecirculation is suspended for a period of time to allow the cells toattach to the substrate. Cells are allowed to attach to side one for 4–6hours; then side two is inoculated and the cells allowed to attach forno more than 18 hours befroe recirculation is restarted. After cellattachment, medium perfusion and recirculation was restarted and thecells were allowed to grow for 7 days at 37° C. under culture conditionsof pH=6.90–7.45, DO=40–50% air saturation. Medium perfusion rate isregulated according to the glucose concentration in the Cellcube™, andwas maintained at between 1–2 g/L. One day before viral infection,medium for perfusion was changed from DMEM+10% FBS to Basal DMEM (noFBS). On day 8, cells were infected with AdCMVp53 virus at amultiplicity of infection (MOI) of 5–50 viral particles per cell basedon 8×10¹⁰ cells total. Medium perfusion was stopped for 1 hr at the timeof infection and then resumed for approximately two days. Mediumrecirculation was maintained throughout the virus infection period.

Virus Harvest and Purification

Previous studies looking at virus release kinetics after Ad5CMV-p53infection of 293 cells determined that maximal virus release from theproducer cells due to the lytic nature of adenovirus was obtained fourto six days after infection. Thus, four to six days after virusinfection, the supernatant from the Cellcube™ modules was removed as apool. The virus supernatant was then clarified by filtration through twoPolyguard 5.0 micron filters, followed by a 5.0 micron Polysep filter(Millipore). The supernatant was then concentrated approximately 10-foldusing tangential flow filtration through a Pellicon cassette (Millipore)of 300 K nominal molecular weight cut-off (NMWC). The buffer was thenexchanged by diafiltration against 0.5 M Tris+1 mM MgCl₂, pH=8. Thesupernatant was then treated at room temperature with 100 U/mlBenzonase™ in a buffer of 0.5M Tris/HCl+1 mM MgCl₂, pH=8.0; 0.2 micronfiltered, and incubated overnight at room temperature to removecontaminating cellular nucleic acids. The crude virus preparation isthen 0.2 micron filtered and loaded directly onto an ion exchange column(BPG 200/500, Pharmacia) containing Source 15Q resin equilibrated with20 mM Tris+1 mM MgCl₂+250 mM NaCl, pH=8.0. The virus was eluted with a40 column linear gradient using an elution buffer composed of 20 mMTris+1 mM MgCl₂+2 M NaCl, pH=8.0. The purified virus was then subjectedto another concentration and diafiltration step to place the virus inthe final formulation for the virus product. The concentration step useda 300 NMWC Pellicon TFF membrane, and for diafiltration the buffer wasexchanged using 8–10 column volumes of Dulbecco's Phosphate BufferedSaline+10% Glycerol. The purified virus was then sterile filteredthrough a 0.2 micron Millipak (Millipore) filter. The formulated productwas then filled into sterile glass vials with stoppers. Flip off crimpcaps were applied prior to final product inspection and labeling.

Two process hold points may be introduced into the process as describedin Example 10. The first process hold may be introduced after the IECstep, at which time 10% glycerol may be added to the eluate and frozenfor later processing. The second process hold step may be introducedafter the final product is obtained but prior to sterile filtering andvialing. The final bulk product can at this point can be frozen and heldfor final filtering and vialing.

The following list of parameters was measured throughout the productionand purification process. The Specification is the desired measurementthat the test article should meet. The result of each test is shown tothe right on the table.

Test Specification Result Mycoplasma PTC Negative PASS 1993 Bioburden≦10 CFU/100 ML 0 CFU/100 ML In Vitro Adventitious NEGATIVE PASS Virus InVivo Adventitious NEGATIVE PASS Virus Adeno-Associated NEGATIVE PASSVirus (PCR) Bioburden ≦1 cfu/10 mL 0 cfu/10 mL Bacterial Endotoxins <5EU/mL <0.15 EU/mL Test Sterility STERILE Pass Sterility Sterile PassBacterial Endotoxins <5 EU/mL <0.15 EU/mL Test Titration of 2 × 10¹⁰–8 ×10¹⁰ 5 × 10¹⁰ pfu/mL Adenovirus Vector pfu/mL Virus Particle 8.0 ×10¹¹–1.2 × 10¹² 9.4 × 10¹¹ Enumeration Viral Particles/mL ViralParticles/mL Ratio 260/280 Ratio 260/280 Particle/pfu Ratio 10–60 20Western Blot Express p53 Protein Pass (anti-p53) Bioactivity (SAOS) MOICausing 50% Cell <1000 vp/cell Death is <1000 vp/cell RestrictionMapping Molecular Size as Pass Expected Protein Content by ≦320 μg/1 ×10¹² 245 μg/1 × 10¹² BCA Viral Particles Viral Particles SDS-PAGE Bandsas Expected Pass No Significant Extra Bands HPLC ≧98% Purity ≧99.57% IonExchange Bovine Serum <50 ng BSA/10¹² <1.9 ng BSA/10¹² Albumin (ELISA)Viral Particles Viral Particles Recoverable Fill 1.0 to 1.4 mL 7 of 7vials in the Volume range of 1.1 to 1.2 mL Physical Description Clear toopalescent with Pass no gross particles by visual inspection huDNA <10ng/1 × 10¹² 0.4 ng/1 × 10¹² Viral Particles Viral Particles GeneralSafety Pass Pass Replication <1 pfu in 2.5 × 10⁹ Viral Report Value atCompetent Particles 2.5 × 10⁹ and Adenovirus 2.5 × 10¹⁰ p53 Mutation <3%<1% Frequency pH 6.0–8.0 7.5

Example 14 Summary of Formulation Development for Adenovirus

Currently, clinical Adp53 product is stored frozen at ≦60° C. This deepfrozen storage condition is not only expensive, but also createsproblems for shipment and inconvenience for clinic use. The goal of theformulation development effort is to develop either a liquid or alyophilized formulation for Adp53 that can be stored at refrigeratedcondition and be stable for extended period of time. Formulationdevelopment for Adp53 is focused on both lyophilization and liquidformulations. From manufacturing and marketing economics point of view,liquid formulation is preferred to a lyophilized formulation.Preliminary results from both fronts of formulation development aresummarized here.

Materials and Equipment

Lyophilizer

A Dura-stop μp lyophilizer (FTSsystems) with in process sampleretrieving device was used. The lyophilizer is equipped with boththermocouple vacuum gauge and capacitance manometer for vacuummeasurement. Condenser temperature is programmed to reach to −80° C.Vials were stoppered at the end of each run with a build-in mechanicalstoppering device.

Residual Moisture Measurement

Residual moisture in freeze dried product was analyzed by a Karl-Fishertype coulometer (Mettler DL37, KF coulometer).

HPLC Analysis

HPLC analysis of samples was done on a Beckman Gold HPLC system.

Vials and Stoppers

Borosilicate 3ml with 13 mm opening lyo vials and their correspondingbutyl rubber stoppers (both from Wheaton) were used for bothlyophilization and liquid formulation development. The stoppered vialswere capped with Flip-off aluminum caps using a capping device (LW312Westcapper, The West Company).

Results

Lyophilization

Initial Cycle and Formulation Development

There are three main process variables that can be programmed to achieveoptimal freeze-drying. Those are shelf temperature, chamber pressure,and lyophilization step duration time. To avoid cake collapse, shelftemperature need to be set at temperatures 2–3° C. below the glasstransition or eutectic temperature of the frozen formulation. Both theglass transition and eutectic temperatures of a formulation can bedetermined by differential scanning coloremetry (DSC) analysis. Chamberpressure is generally set at below the ice vapor pressure of the frozenformulation. The ice vapor pressure is dependent on the shelftemperature and chamber pressure. Too high a chamber pressure willreduce the drying rate by reducing the pressure differential between theice and the surrounding, while too low a pressure will also slow downdrying rate by reducing the heat transfer rate from the shelf to thevials. The development of a lyophilization cycle is closely related withthe formulation and the vials chosen for lyophilization. Formulationexcipient selection was based on the classical expcipients found in mostlyophilized pharmaceuticals. The excipients in a lyophilizationformulation should provide the functions of bulking, cryoprotection, andlyoprotection. The excipients chosen were mannitol (M, bulking agent),sucrose (S, cryo- and lyoprotectant), and human serum albumin (HSA,lyoprotectant). These excipients were formulated in 10 mM Tris+1 mMMgCl₂, pH=7.50 at various percentages and filled into the 3 ml vials ata fill volume of 1 ml. To start with, a preliminary cycle was programmedto screen a variety of formulations based on the criteria of residualmoisture and physical appearance after drying. The cycle used is plottedin FIG. 29. Extensive screening was carried out by variation of thepercentages of the individual excipients. Table 18 shows briefly some ofthe results.

TABLE 18 Evaluation of different formulations under the same cycleFormulation M %/S %/HSA % Appearance Moisture (% weight) 10/5/0.5 goodcake 0.89 5/5/0.5 good cake 1.5 3/5/0.5 loose cake 3.4 (partialcollapse) 1/5/0.5 no cake (collapse) 6.4

The results suggest that a minimum amount of 3% mannitol is required inthe formulation in order to achieve pharmaceutically elegant cake. Thepercentages of sucrose in the formulation were also examined. Nosignificant effect on freeze-drying was observed at sucroseconcentrations of ≦10%. HSA concentration was kept constant to 0.5%during the initial screening stage.

After the evaluation of the formulations, freeze-drying cycle wasoptimized by changing the shelf temperature, chamber vacuum and theduration of each cycle step. Based on the extensive cycle optimization,the following cycle (cycle #14) was used for further viruslyophilization development.

-   -   Load sample at room temperature onto shelf    -   Set shelf temperature to −45° C. and freeze sample. Step time 2        h.    -   Set shelf temperature at −45° C., turn vacuum pump and set        vacuum at 400 mT. Step time 5 h    -   Set shelf temperature at −35° C., set vacuum at 200 mT. Step        time 13 h    -   Set shelf temperature at −22° C., set vacuum at 100 mT. Step        time 15 h    -   Set shelf temperature at −10° C., set vacuum at 100 mT. Step        time 5 h    -   Set shelf temperature at 10° C., set vacuum at 100 mT, Step time        4 h    -   Vial stoppering under vacuum

Cycle and Formulation Development with Virus in Formulation

Effect of Sucrose Concentration in Formulation

Cycle and formulation were further optimized according to virus recoveryafter lyophilization analyzed by both HPLC and plaque forming unit (PFU)assays. Table 19 shows the virus recoveries immediate after drying indifferent formulations using the above drying cycle. Variation of thepercentage of sucrose in the formulation had significant effect on virusrecoveries.

TABLE 19 Recoveries of virus after lyophilization Formulation Residual M%/S %/HSA % Appearance moisture Recovery (%) 6/0/0.5 Good cake  0.44%  06/3.5/0.5 Good cake 2.2%  56 6/5/0.5 Good cake 2.5%  81 6/6/0.5 Goodcake 2.7% 120 6/7/0.5 Good cake 2.8% 120 6/8/0.5 Good cake 3.3%  936/9/0.5 Good cake 3.7% 120

Residual moisture in the freeze-dried product increased as the sucrosepercentage increased. A minimum sucrose concentration of 5% is requiredin the formulation to maintain a good virus recovery afterlyophilization. Similar sucrose effects in formulation that had 5%instead of 6% mannitol were observed. However, good virus recoveryimmediately after drying does not necessary support a good long-termstorage stability. As a result, formulations having 4 different sucroseconcentrations of 6, 7, 8 and 9%, were incorporated for furtherevaluation.

Effect of HSA in Formulation

The contribution of HSA concentrations in the formulation on virusrecovery after drying was examined using the same freeze drying cycle.Table 20 shows the results

TABLE 20 Effects of HSA concentration on lyophilization FormulationResidual Recovery M %/S %/HSA % Appearance moisture (%) 6/7/0 Good cake0.98  83 6/7/0.5 Good cake 1.24 120 6/7/2 Good cake 1.5 110 6/7/5 Goodcake 1.7 102

The results indicate that inclusion of HSA in the formulation hadpositive effect on virus recovery after drying. Concentrations higherthan 0.5% did not further improve the virus recovery post drying. As aresult, 0.5% HSA is formulated in all the lyophilization formulations.

Cycle Optimization

As indicated in Table 19, relatively high residual moistures werepresent in the dried product. Although there has not been a knownoptimal residual moisture for freeze dried viruses, it could bebeneficial for long term storage stability to further reduce theresidual moisture in the dried product. After reviewing of the dryingcycle, it was decided to increase the secondary drying temperature from10° C. to 30° C. without increasing the total cycle time. As indicatedin Table 21, significant reduction in residual moisture had beenachieved in all the formulations without negative effects on virusrecoveries. With the improved drying cycle, residual moisture was lessthan 2% in all the formulations immediately after drying. It is expectedthat the reduced residual moisture will improve the long-term storagestability of the dried product.

TABLE 21 Effects of secondary drying temperature on lyophilizationSecondary Secondary drying at 10° C. drying at 30° C. FormulationResidual Recovery Residual M %/S %/HAS % moisture (w %) (%) moistureRecovery 6/6/0.5 2.2 100 0.8 93 6/7/0.5 2.5  86 1.1 100  6/8/0.5 2.7  831.3 87 6/9/0.5 3.3  93 1.5 86 5/6/0.5 2.3 110 1.0 94 5/7/0.5 2.7  88 1.285 5/8/0.5 3.5  97 1.6 88 5/9/0.5 4  90 1.9 86

N₂ Backfilling (Blanketing)

Lyophilization was done similarly as above except that dry N₂ was usedfor gas bleeding for pressure control during the drying and backfillingat the end of the cycle. At the end of a drying run, the chamber wasfilled with dry N₂ to about 80% atmospheric pressure. Subsequently, thevials were stoppered. No difference was noticed between the air and N₂blanketing runs immediate after drying. However, if oxygen present inthe vial during air backfilling causes damaging effect (oxidation) onthe virus or excipients used during long-term storage, backfilling withdry N₂ is likely to ameliorate the damaging effects and improve longterm storage stability of the virus.

Removal of Glycerol from Formulation

During the preparation of virus containing formulations, stock virussolution was added to the pre-formulated formulations at a dilutionfactor of 10. Because of the presence of 10% glycerol in the stock virussolution, 1% glycerol was introduced into the formulations. To examineany possible effect of the presence of 1% glycerol on lyophilization, afreeze drying run was conducted using virus diafiltered into theformulation of 5% (M)/7%(S)/0.5% (HSA). Diafiltration was done with 5vol of buffer exchange using a constant volume buffer exchange mode toensure adequate removal of residual glycerol (99% removal). Afterdiafiltration, virus solution was filled into vials and then lyophilizedsimilarly. Table 22 shows the lyophilization results

TABLE 22 Lyophilization without glycerol Formulation M %/S %/HSA %Residual moisture Recovery (%) 5/7/0.5 1.0 80

No significant difference after freeze drying was observed betweenformulations with and without 1% glycerol. Possible implications of thischange on long term storage will be evaluated.

Long Term Storage Stability

Adp53 virus lyophilized under different formulations and differentcycles was placed at −20° C., 4° C., and room temperature (RT) underdark for long term storage stability evaluation. Parameters measuredduring the stability study were PFU, HPLC viral particles, residualmoisture, and vacuum inside vial (integrity). FIG. 30A and FIG. 30B showthe data after 12-month storage with secondary drying at 10° C. withoutN₂ blanketing. Lyophilized virus is stable at both −20° C. and 4° C.storage for up to 12 months. However, virus was not stable at roomtemperature storage. More than 50% loss in infectivity was observed atRT after 1-month storage. The reason for the quick loss of infectivityat RT is not clear. However, it is likely that RT is above the glasstransition temperature of the dried formulation and results in theaccelerated virus degradation. A differential scanning colorimitry (DSC)analysis of the formulation could provide very useful information.Pressure change inside the vials during storage was not detected, whichindicates that the vials maintained their integrity. The slight increasein residual moisture during storage can be attributed to the release ofmoisture from the rubber stopper into the dried product.

FIG. 31 and FIG. 32 show the storage stability data with secondarydrying at 30° C. without and with N₂ backfilling, respectively. Becauseof the nearly identical stability observed at −20° C. and 4° C. storageconditions, and to reduce the consumption of virus, −20° C. was notincluded in the long-term storage stability study. Similar to thesamples dried with secondary drying at 10° C., virus is stable at 4° C.but not stable at RT. However, relative better stability was observed atRT storage than those dried at 10° C. secondary drying. This is likelyto be the result of the lower residual moisture attained at 30° C.secondary drying. This result suggests that residual moisture is animportant parameter that affects storage stability during long termstorage. Longer time storage is needed to reveal any beneficial effectsof doing N₂ blanketing during lyophilization since no significant effectwas observed for up to 3 months storage. During storage, HPLC analysisindicates that virus is stable at both −20° C. and 4° C. storage and notstable at RT, which is consistent with the results from PFU assay.

HSA Alternatives

The presence of HSA in the formulations could be a potential regulatoryconcern. As a result, a variety of excipients have been evaluated tosubstitute HSA in the formulation.

The substitutes examined included PEG, amino acids (glycine, arginine),polymers (polyvinylpyrrolidone), and surfactants (Tween-20 andTween-80).

Liquid Formulation

Concurrent with the development of lyophilization of Adp53 product,experimentation was carried out to examine the possibility of developinga liquid formulation for Adp53 product. The goal was to develop aformulation that can provide enough stability to the virus when storedat above freezing temperatures. Four sets of liquid formulations havebeen evaluated. In the first set of formulation, the current 10%glycerol formulation was compared to HSA and PEG containingformulations. In the second set of formulation, various amino acids wereexamined for formulating Adp53. In the third set of formulation, theoptimal formulation developed for lyophilization was used to formulateAdp53 in a liquid form. In the fourth set of formulation, detergentswere evaluated for formulating Adp53. Viruses formulated with all thosedifferent formulations are being tested for long term storage stabilityat −20° C., 4° C., and RT.

Liquid Formulation set #1

HSA containing formulation (5% sucrose+5%HSA in 10 mM Tris buffer, 150mM NaCl, and 1 mM MgCl₂, pH=8.20 buffer) was compared with 10% glycerolin DPBS buffer and sucrose/PEG and Trehalose/PEG formulations. PEG hasbeen recommended as a good preferential exclusion agent in formulations(Wong and Parasrampurita, Parmaceutical excipients for the stabilizationof proteins, BioPharm, 10(11) 52–61, 1997). It is included in this setof formulation to examine whether it can provide stabilization effect onAdp53. Formulations were filled into the 3 ml lyo vials at a fill volumeof 0.5 ml. Vials were capped under either atmospheric or N₂ blanketingconditions to examine any positive effects N₂ blanketing may have onlong term storage stability of Adp53. To ensure adequate degassing fromthe formulation and subsequent N₂ blanketing, the filled vials waspartially stoppered with lyo stoppers and loaded onto the shelf of thelyophilizer under RT. The lyophilizer chamber was closed and vacuum wasestablished by turning on the vacuum pump. The chamber was evacuated to25 in. Hg. Then the chamber was purged completely with dry N₂. Theevacuation and gassing were repeated twice to ensure complete N₂blanketing. N₂ blanketed vials were placed with the non-N₂ blanketedvials at various storage conditions for storage stability evaluation.FIG. 33 shows the analysis data for upto 9 months storage at 4° C. andRT.

Statistically significant drops in virus PFU and HPLC viral particleswere observed for 10% glycerol formulation after 3 months storage atboth 4° C. and RT. No statistically significant virus degradation wasobserved for all other formulations at 4° C. storage. However, decreasein virus infectivity was observed when stored at RT. Longer time storageis needed to evaluate the effectiveness of the different formulations.

Liquid Formulation #2

Various combinations of amino acids, sugars, PEG and urea were evaluatedfor Adp53 stabilization during long storage. FIG. 34 shows the 6-monthstability data. The results indicate that combination of 5% mannitol and5% sucrose with other excipients gave better storage stability at RT. Inthis set of formulation, no human or animal derived excipients wereincluded.

Liquid Formulation set #3

The optimal formulations developed for lyophilization was evaluated forformulating Adp53 in a liquid form. This approach would be a goodbridging between liquid formulation and lyophilization if satisfactoryAdp53 stability can be achieved using lyophilization formulation forliquid fill. Filled samples were stored at −20° C. and 4° C. forstability study. FIG. 35 shows the 3-month stability data. Virus isstable at both −20° C. and 4° C. for the four different formulations.This is in agreement with the results from formulation set #2, whichsuggests that better virus stability is expected with the presence ofboth mannitol and sucrose in the formulation. Longer time storagestability data is being accrued.

Liquid Formulation set #4

Detergents have been used in the formulations for a variety ofrecombinant proteins. In this set of formulation, various concentrationsof detergents were examined for formulating Adp53. The detergents usedwere non-ionic (Tween-80) and zwitterionic (Chaps). FIG. 36 shows the6-month stability data. Virus is stable at 4° C. storage. Better virusstability is observed in Tween-80 containing formulations. Furtheraccumulation of stability data will help to optimize the detergentconcentration. Similar to formulation set#2, no exogenous protein isincluded in this set of formulations.

Both lyophilization and liquid formulation have produced veryinteresting and promising data and information. A lyophilization cycleand corresponding formulations have been developed to producelyophilized Adp53 that is stable at 4° C. for at least 12 months. Longertime storage stability is being collected. Because of the conservativeapproach taken in the initial development of the lyophilization cycle,we are investigating further to significantly reduce the lyophilizationcycle time and to improve the lyophilization process efficiency.Somewhat to our surprise, very promising stability data was generatedfor liquid formulation at 4° C. storage. However, longer time storagedata is needed to evaluate the feasibility of developing a liquidformulation for Adp53.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A recombinant adenovirus composition comprising between 5×10¹⁴ and1×10¹⁸ viral particles and having less than 50 ng of BSA per 1×10¹²viral particles.
 2. A purified recombinant adenovirus compositioncomprising between 5×10¹⁴ and 1×10¹⁸ adenoviral particles and betweenabout 100 pg and 10 ng of contaminating human DNA per 1×10¹² viralparticles.
 3. The purified recombinant adenovirus composition of claim 1or 2, said composition having between about 100 pg and 10 ng ofcontaminating human DNA per 1×10¹² viral particles and one or more ofthe following properties: (a) a virus titer of between about 1×10⁹ andabout 1×10¹³ pfu/ml; (b) a virus particle concentration between about1×10¹⁰ and about 2×10¹³ particles/ml; (c) a particle:pfu ratio betweenabout 10 and about 60; (d) having less than 50 ng BSA per 1×10¹² viralparticles; (e) elutes essentially as a single peak upon HPLC.
 4. Thecomposition of claim 1 or 2, wherein the composition has a virus titerof between about 1×10¹¹ and about 1×10¹³ pfu/ml.
 5. The composition ofclaim 4, wherein the composition has a virus titer of between about1×10¹² and about 1×10¹³ pfu/ml.
 6. The composition of claim 1 or 2,wherein the composition has a virus particle concentration between about1×10¹¹ and about 2×10¹³ particles/ml.
 7. The composition of claim 6,wherein the composition has a virus particle concentration between about1×10¹² and about 1×10¹³ particles/ml.
 8. The composition of claim 1 or2, wherein the composition has a particle:pfu ratio between about 10 andabout
 50. 9. The composition of claim 8, wherein the composition has aparticle:pfu ratio between about 10 and about
 40. 10. The composition ofclaim 9, wherein the composition has a particle:pfu ratio between about20 and about
 40. 11. The composition of claim 1 or 2, wherein thecomposition has between about 1 ng and 50 ng of BSA per 1×10¹² viralparticles.
 12. The composition of claim 11, wherein the composition hasbetween about 5 ng and 40 ng BSA per 1×10¹² viral particles.
 13. Thecomposition of claim 1 or 2, wherein the composition has between about100 pg and 500 pg of contaminating human DNA per 1×10¹² viral particles.14. The composition of claim 1 or 2, wherein the adenovirus of saidcomposition elutes as essentially a single HPLC peak that comprisesbetween 97 and 99% of the total area under the peak.
 15. The compositionof claim 1 or 2, wherein the composition has between about 100 pg andabout 7 ng of contaminating human DNA per 10×10¹² viral particles. 16.The composition of claim 15, wherein the composition has between about100 pg and about 5 ng of contaminating human DNA per 1×10¹² viralparticles.
 17. The composition of claim 16, wherein the composition hasbetween about 100 pg and about 3 ng of contaminating human DNA per1×10¹² viral particles.
 18. The composition of claim 17, wherein thecomposition has between about 100 pg and about 1 ng of contaminatinghuman DNA per 1×10¹² viral particles.
 19. The composition of claim 17,wherein the composition has between about 100 pg and about 500 pg ofcontaminating human DNA per 1×10¹² viral particles.
 20. The compositionof claim 3, wherein the composition has all of (a)–(e).